U.S. patent application number 10/240842 was filed with the patent office on 2003-06-12 for regulation of human hm74-like g protein coupled receptor.
Invention is credited to Yonghong, Xiao.
Application Number | 20030109673 10/240842 |
Document ID | / |
Family ID | 22908165 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109673 |
Kind Code |
A1 |
Yonghong, Xiao |
June 12, 2003 |
Regulation of human hm74-like g protein coupled receptor
Abstract
Reagents which regulate human HM74-like G protein coupled
receptor can play a role in preventing, ameliorating, or correcting
dysfunctions or diseases including, but not limited to, infections
such as bacterial, fungal, protozoan, and viral infections,
particularly those caused by HIV viruses, pain, cancers, anorexia,
bulimia, asthma, CNS diseases such as Parkinson's disease,
cardiovascular diseases such as acute heart failure, hypotension,
hypertension, angina pectoris, and myocardial infarction, urinary
retention, osteoporosis, ulcers, asthma, inflammation, allergies,
multiple sclerosis, benign prostatic hypertrophy, and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, several mental retardation, and
dyskinesias, such as Huntington's disease and Tourett's
syndrome.
Inventors: |
Yonghong, Xiao; (Cambridge,
MA) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
22908165 |
Appl. No.: |
10/240842 |
Filed: |
October 4, 2002 |
PCT Filed: |
April 4, 2001 |
PCT NO: |
PCT/EP01/03811 |
Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 536/23.5 |
Current CPC
Class: |
C07K 14/705
20130101 |
Class at
Publication: |
530/350 ;
435/69.1; 435/320.1; 435/325; 536/23.5 |
International
Class: |
C07K 014/705; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
1. An isolated polynucleotide encoding a HM74-like GPCR polypeptide
and being selected from the group consisting of: a) a
polynucleotide encoding a HM74-like GPCR polypeptide comprising an
amino acid sequence selected from the group consisting of: amino
acid sequences which are at least about 53% identical to the amino
acid sequence shown in SEQ ID NO: 2; and the amino acid sequence
shown in SEQ ID NO: 2. b) a polynucleotide comprising the sequence
of SEQ ID NO: 1; c) a polynucleotide which hybridizes under
stringent conditions to a polynucleotide specified in (a) and (b);
d) a polynucleotide the sequence of which deviates from the
polynucleotide sequences specified in (a) to (c) due to the
degeneration of the genetic code; and e) a polynucleotide which
represents a fragment, derivative or allelic variation of a
polynucleotide sequence specified in (a) to (d).
2. An expression vector containing any polynucleotide of claim
1.
3. A host cell containing the expression vector of claim 2.
4. A substantially purified HM74-like GPCR polypeptide encoded by a
polynucleotide of claim 1.
5. A method for producing a HM74-like GPCR polypeptide, wherein the
method comprises the following steps: a) culturing the host cell of
claim 3 under conditions suitable for the expression of the
HM74-like GPCR polypeptide; and b) recovering the HM74-like GPCR
polypeptide from the host cell culture.
6. A method for detection of a polynucleotide encoding a HM74-like
GPCR polypetide in a biological sample comprising the following
steps: a) hybridizing any polynucleotide of claim 1 to a nucleic
acid material of a biological sample, thereby forming a
hybridization complex; and b) detecting said hybridization
complex.
7. The method of claim 6, wherein before hybridization, the nucleic
acid material of the biological sample is amplified.
8. A method for the detection of a polynucleotide of claim 1 or a
HM74-like GPCR polypeptide of claim 4 comprising the steps of
contacting a biological sample with a reagent which specifically
interacts with the polynucleotide or the HM74-like GPCR polypeptide
and detecting the interaction.
9. A diagnostic kit for conducting the method of any one of claims
6 to 8.
10. A method of screening for agents which decrease the activity of
a HM74-like GPCR, comprising the steps of: contacting a test
compound with any HM74-like GPCR polypeptide encoded by any
polynucleotide of claim 1; detecting binding of the test compound
of the HM74-like GPCR polypeptide, wherein a test compound which
binds to the polypeptide is identified as a potential therapeutic
agent for decreasing the activity of a HM74-like GPCR.
11. A method of screening for agents which regulate the activity-of
a EM74-like GPCR, comprising the steps of: contacting a test
compound with a HM74-like GPCR polypeptide encoded by any
polynucleotide of claim 1; and detecting a HM74-like GPCR activity
of the polypeptide, wherein a test compound which increases the
HM74-like GPCR activity is identified as a potential therapeutic
agent for increasing the activity of the HM74-like GPCR, and
wherein a test compound which decreases the HM74-like GPCR activity
of the polypeptide is identified as a potential therapeutic agent
for decreasing the activity of the HM74-like GPCR.
12. A method of screening for agents which decrease the activity of
a HM74-like GPCR, comprising the steps of: contacting a test
compound with any polynucleotide of claim 1 and detecting binding
of the test compound to the polynucleotide, wherein a test compound
which binds to the polynucleotide is identified as a potential
therapeutic agent for decreasing the activity of HM74-like
GPCR.
13. A method of reducing the activity of HM74-like GPCR, comprising
the steps of: contacting a cell with a reagent which specifically
binds to any polynucleotide of claim 1 or any HM74-like GPCR
polypeptide of claim 4, whereby the activity of HM74-like GPCR is
reduced.
14. A reagent that modulates the activity of a HM74-like GPCR
polypeptide or a polynucleotide wherein said reagent is identified
by the method of any of the claims 10 to 12.
15. A pharmaceutical composition, comprising: the expression vector
of claim 2 or the reagent of claim 14 and a pharmaceutically
acceptable carrier.
16. Use of the pharmaceutical composition of claim 15 for
modulating the activity of a HM74-like GPCR in a disease.
17. Use of claim 16 wherein the disease is bacterial, fungal,
protozoan, and viral infection, pain, cancer, anorexia, bulimia,
asthma, CNS disease, cardiovascular disease, hypotension,
hypertension, angina pectoris, and myocardial infarction, urinary
retention, osteoporosis, ulcer, asthma, inflammation, allergy,
multiple sclerosis, benign prostatic hypertrophy, and psychotic and
neurological disorder, and dyskinesia.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to the area of G-protein coupled
receptors. More particularly, it relates to the area of HM74-like G
protein coupled receptors and their regulation.
BACKGROUND OF THE INVENTION
[0002] G-Protein Coupled Receptors
[0003] Many medically significant biological processes are mediated
by signal transduction pathways that involve G-proteins (Lefkowitz,
Nature 351, 353-354, 1991). The family of G-protein coupled
receptors (GPCR) includes receptors for hormones,
neurotransmitters, growth factors, and viruses. Specific examples
of GPCRs include receptors for such diverse agents as dopamine,
calcitonin, adrenergic hormones, endothelin, cAMP, adenosine,
acetylcholine, serotonin, histamine, thrombin, kinin, follicle
stimulating hormone, opsins, endothelial differentiation gene-1,
rhodopsins, odorants, cytomegalovirus, G-proteins themselves,
effector proteins such as phospholipase C, adenyl cyclase, and
phosphodiesterase, and actuator proteins such as protein kinase A
and protein kinase C.
[0004] GPCRs possess seven conserved membrane-spanning domains
connecting at least eight divergent hydrophilic loops. GPCRs (also
known as 7TM receptors) have been characterized as including these
seven conserved hydrophobic stretches of about 20 to 30 amino
acids, connecting at least eight divergent hydrophilic loops. Most
GPCRs have single conserved cysteine residues in each of the first
two extracellular loops, which form disulfide bonds that are
believed to stabilize functional protein structure. The seven
transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5,
TM6, and TM7. TM3 has been implicated in signal transduction.
[0005] Phosphorylation and lipidation (palmitylation or
farnesylation) of cysteine residues can influence signal
transduction of some GPCRs. Most GPCRs contain potential
phosphorylation sites within the third cytoplasmic loop and/or the
carboxy terminus. For several GPCRs, such as the .beta.-adrenergic
receptor, phosphorylation by protein kinase A and/or specific
receptor kinases mediates receptor desensitization.
[0006] For some receptors, the ligand binding sites of GPCRs are
believed to comprise hydrophilic sockets formed by several GPCR
transmembrane domains. The hydrophilic sockets are surrounded by
hydrophobic residues of the GPCRs. The hydrophilic side of each
GPCR transmembrane helix is postulated to face inward and form a
polar ligand binding site. TM3 has been implicated in several GPCRs
as having a ligand binding site, such as the TM3 aspartate residue.
TM5 serines, a TM6 asparagine, and TM6 or TM7 phenylalanines or
tyrosines also are implicated in ligand binding.
[0007] GPCRs are coupled inside the cell by heterotrimeric
G-proteins to various intracellular enzymes, ion channels, and
transporters (see Johnson et al., Endoc. Rev. 10, 317-331, 1989).
Different G-protein alpha-subunits preferentially stimulate
particular effectors to modulate various biological functions in a
cell. Phosphorylation of cytoplasmic residues of GPCRs is an
important mechanism for the regulation of some GPCRs. For example,
in one form of signal transduction, the effect of hormone binding
is the activation inside the cell of the enzyme, adenylate cyclase.
Enzyme activation by hormones is dependent on the presence of the
nucleotide GTP. GTP also influences hormone binding. A G-protein
connects the hormone receptor to adenylate cyclase. G-protein
exchanges GTP for bound GDP when activated by a hormone receptor.
The GTP-carrying form then binds to activated adenylate cyclase.
Hydrolysis of GTP to GDP, catalyzed by the G-protein itself,
returns the G-protein to its basal, inactive form. Thus, the
G-protein serves a dual role, as an intermediate that relays the
signal from receptor to effector, and as a clock that controls the
duration of the signal.
[0008] Over the past 15 years, nearly 350 therapeutic agents
targeting GPCRs receptors have been successfully introduced onto
the market. This indicates that these receptors have an
established, proven history as therapeutic targets. Clearly, there
is an on-going need for identification and characterization of
further GPCRs which can play a role in preventing, ameliorating, or
correcting dysfunctions or diseases including, but not limited to,
infections such as bacterial, fungal, protozoan, and viral
infections, particularly those caused by HIV viruses, pain,
cancers, anorexia, bulimia, asthma, CNS diseases such as
Parkinson's disease, cardiovascular diseases such as acute heart
failure, hypotension, hypertension, angina pectoris, and myocardial
infarction, urinary retention, osteoporosis, ulcers, asthma,
inflammation, allergies, multiple sclerosis, benign prostatic
hypertrophy, and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
several mental retardation, and dyskinesias, such as Huntington's
disease and Tourett's syndrome.
[0009] Because of the wide-spread distribution of GPCRs with
diverse biological effects, there is a need in the art to identify
additional members of the GPCR family whose activity can be
regulated to provide therapeutic effects.
SUMMARY OF THE INVENTION
[0010] It is an object of the invention to provide reagents and
methods of regulating a human HM74-like G protein coupled receptor
(HM74-like GPCR). This and other objects of the invention are
provided by one or more of the embodiments described below.
[0011] One embodiment of the invention is a HM74-like GPCR
polypeptide comprising an amino acid sequence selected from the
group consisting of:
[0012] amino acid sequences which are at least about 53% identical
to the amino acid sequence shown in SEQ ID NO: 2; and
[0013] the amino acid sequence shown in SEQ ID NO: 2.
[0014] Yet another embodiment of the invention is a method of
screening for agents which decrease the activity of HM74-like GPCR.
A test compound is contacted with a HM74-like GPCR polypeptide
comprising an amino acid sequence selected from the group
consisting of:
[0015] amino acid sequences which are at least about 53% identical
to the amino acid sequence shown in SEQ ID NO: 2; and
[0016] the amino acid sequence shown in SEQ ID NO: 2.
[0017] Binding between the test compound and the HM74-like GPCR
polypeptide is detected. A test compound which binds to the
HM74-like GPCR polypeptide is thereby identified as a potential
agent for decreasing the activity of HM74-like GPCR.
[0018] Another embodiment of the invention is a method of screening
for agents which decrease the activity of HM74-like GPCR. A test
compound is contacted with a polynucleotide encoding a HM74-like
GPCR polypeptide, wherein the polynucleotide comprises a nucleotide
sequence selected from the group consisting of:
[0019] nucleotide sequences which are at least about 53% identical
to the nucleotide sequence shown in SEQ ID NO: 1; and
[0020] the nucleotide sequence shown in SEQ ID NO: 1.
[0021] Binding of the test compound to the polynucleotide is
detected. A test compound which binds to the polynucleotide is
identified as a potential agent for decreasing the activity of
HM74-like GPCR. The agent can work by decreasing the amount of the
HM74-like GPCR through interacting with the HM74-like GPCR
mRNA.
[0022] Another embodiment of the invention is a method of screening
for agents which regulate the activity of HM74-like GPCR. A test
compound is contacted with a HM74-like GPCR polypeptide comprising
an amino acid sequence selected from the group consisting of:
[0023] amino acid sequences which are at least about 53% identical
to the amino acid sequence shown in SEQ ID NO: 2; and
[0024] the amino acid sequence shown in SEQ ID NO: 2.
[0025] A HM74-like GPCR activity of the polypeptide is detected. A
test compound which increases HM74-like GPCR activity of the
polypeptide relative to HM74-like GPCR activity in the absence of
the test compound is thereby identified as a potential agent for
increasing the activity of HM74-like GPCR. A test compound which
decreases HM74-like GPCR activity of the polypeptide relative to
HM74-like GPCR activity in the absence of the test compound is
thereby identified as a potential agent for decreasing the activity
of HM74-like GPCR.
[0026] Even another embodiment of the invention is a method of
screening for agents which decrease the activity of HM74-like GPCR.
A test compound is contacted with a HM74-like GPCR product of a
polynucleotide which comprises a nucleotide sequence selected from
the group consisting of:
[0027] nucleotide sequences which are at least about 53% identical
to the nucleotide sequence shown in SEQ ID NO: 1; and
[0028] the nucleotide sequence shown in SEQ ID NO: 1.
[0029] Binding of the test compound to the HM74-like GPCR product
is detected. A test compound which binds to the HM74-like GPCR
product is thereby identified as a potential agent for decreasing
the activity of HM74-like GPCR.
[0030] Still another embodiment of the invention is a method of
reducing the activity of HM74-like GPCR. A cell is contacted with a
reagent which specifically binds to a polynucleotide encoding a
HM74-like GPCR polypeptide or the product encoded by the
polynucleotide, wherein the polynucleotide comprises a nucleotide
sequence selected from the group consisting of:
[0031] nucleotide sequences which are at least about 53% identical
to the nucleotide sequence shown in SEQ ID NO: 1; and
[0032] the nucleotide sequence shown in SEQ ID NO: 1.
[0033] HM74-like GPCR activity in the cell is thereby
decreased.
[0034] The invention thus provides an HM74-like G protein coupled
receptor which can be used to identify test compounds which may act
as agonists or antagonists at the receptor site and which can be
regulated to provide therapeutic effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows the DNA-sequence encoding a HM74-like GPCR
polypeptide.
[0036] FIG. 2 shows the amino acid sequence deduced from the
DNA-sequence of FIG. 1.
[0037] FIG. 3 shows the amino acid sequence of a part of the
protein Assigned Swissprot Accession No. P49019.
[0038] FIG. 4 shows an alignment of HM74-like GPCR polypeptide and
the HM74 protein having Swissprot Accession No. P49019 (SEQ ID NO:
3). Bold indicates transmembrane helix. Underlined region is a GPCR
region Identified using the Prosite database.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The invention relates to an isolated polynucleotide encoding
a HM74-like GPCR polypeptide and being selected from the group
consisting of:
[0040] a) a polynucleotide encoding a HM74-like GPCR polypeptide
comprising an amino acid sequence selected from the group
consisting of:
[0041] amino acid sequences which are at least about 53% identical
to
[0042] the amino acid sequence shown in SEQ ID NO: 2; and
[0043] the amino acid sequence shown in SEQ ID NO: 2;
[0044] b) a polynucleotide comprising the sequence of SEQ ID NO:
1;
[0045] c) a polynucleotide which hybridizes under stringent
conditions to a polynucleotide specified in (a) and (b);
[0046] d) a polynucleotide the sequence of which deviates from the
polynucleotide sequences specified in (a) to (c) due to the
degeneration of the genetic code; and
[0047] e) a polynucleotide which represents a fragment, derivative
or allelic variation of a polynucleotide sequence specified in (a)
to (d).
[0048] Furthermore, it has been discovered by the present applicant
that a HM74-like GPCR, particularly a human HM74-like GPCR, can be
used in therapeutic methods to treat disorders such as bacterial,
fungal, protozoan, and viral infections, particularly those caused
by HIV viruses, pain, cancers, anorexia, bulimia, asthma,
cardiovascular diseases such as acute heart failure, hypotension,
hypertension, angina pectoris, and myocardial infarction, urinary
retention, osteoporosis, inflammation, ulcers, asthma, allergies,
multiple sclerosis, benign prostatic hypertrophy, and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, several mental retardation, and
dyskinesias, such as Parkinson's disease, Huntington's disease, and
Tourett's syndrome. Human HM74-like GPCR also can be used to screen
for human HM74-like GPCR agonists and antagonists.
[0049] An amino acid sequence of a human HM74-like GPCR is shown in
SEQ ID NO: 2. Transmembrane helices are present from amino acids
20-37, 53-73, 91-113, 133-150, 180-197, 223-242, and 260-279 of SEQ
ID NO: 2. A GPCR region is detected between amino acids 101-118 of
SEQ ID NO: 2. Using the BLASTP alignment algorithm, the amino acid
sequence shown in SEQ ID NO: 2 is 52% identical over 341 amino
acids to the protein assigned Swissprot Accession No. P49019 (SEQ
ID NO: 3) and annotated as "probable G protein-coupled receptor
HM74." Human HM74-like GPCR is therefore expected to bind a ligand
to produce a biological effect. or activity typical of GPCRs, such
as cyclic AMP formation, mobilization of intracellular calcium, or
phosphoinositide metabolism.
[0050] Polypeptides
[0051] HM74-like GPCR polypeptides according to the invention
comprise the amino acid sequence shown in SEQ ID NO: 2, a portion
of that sequence comprising 15, 20, 25, or 50 or more contiguous
amino acids, or a biologically active variant thereof, as defined
below. An HM74-like GPCR polypeptide of the invention therefore can
be a portion of an HM74-like GPCR, a full-length HM74-like GPCR, or
a fusion protein comprising all or a portion of an HM74-like
GPCR.
[0052] Biologically Active Variants
[0053] HM74-like GPCR polypeptide variants which are biologically
active, i.e., retain the ability to bind a ligand to produce a
biological effect, such as cyclic AMP formation, mobilization of
intracellular calcium, or phosphoinositide metabolism, also are
HM74-like GPCR polypeptides. Preferably, naturally or non-naturally
occurring HM74-like GPCR polypeptide variants have amino acid
sequences which are at least about 53, preferably about 75, 90, 96,
or 98% identical to an amino acid sequence shown in SEQ ID NO: 2 or
a fragment thereof Percent identity between a putative HM74-like
GPCR polypeptide variant and an amino acid sequence of SEQ ID NO: 2
is determined using the Blast2 alignment program.
[0054] Variations in percent identity can be due, for example, to
amino acid substitutions, insertions, or deletions. Amino acid
substitutions are defined as one for one amino acid replacements.
They are conservative in nature when the substituted amino acid has
similar structural and/or chemical properties. Examples of
conservative replacements are substitution of a leucine with an
isoleucine or valine, an aspartate with a glutamate, or a threonine
with a serine.
[0055] Amino acid insertions or deletions are changes to or within
an amino acid sequence. They typically fall in the range of about 1
to 5 amino acids. Guidance in determining which amino acid residues
can be substituted, inserted, or deleted without abolishing
biological or immunological activity of an HM74-like GPCR
polypeptide can be found using computer programs well known in the
art, such as DNASTAR software. Whether an amino acid change results
in a biologically active HM74-like GPCR polypeptide can readily be
determined by assaying for binding to a ligand or by conducting a
functional assay, such as those described in the specific examples,
below.
[0056] Fusion Proteins
[0057] Fusion proteins are useful for generating antibodies against
HM74-like GPCR polypeptide amino acid sequences and for use in
various assay systems. For example, fusion proteins can be used to
identify proteins which interact with portions of an HM74-like GPCR
polypeptide. Protein affinity chromatography or library-based
assays for protein-protein interactions, such as the yeast
two-hybrid or phage display systems, can be used for this purpose.
Such methods are well known in the art and also can be used as drug
screens.
[0058] An HM74-like GPCR polypeptide fusion protein comprises two
polypeptide segments fused together by means of a peptide bond. The
first polypeptide segment comprises at least 15, 20, 25, or 50 or
more contiguous amino acids of SEQ ID NO: 2 or a biologically
active variant thereof such as those described above. The first
polypeptide segment also can comprise full-length HM74-like
GPCR.
[0059] The second polypeptide segment can be a full-length protein
or a protein fragment. Proteins commonly used in fusion protein
construction include .beta.-galactosidase, .beta.-glucuronidase,
green fluorescent protein (GFP), autofluorescent proteins,
including blue fluorescent protein (BFP), glutathione-S-transferase
(GST), luciferase, horseradish peroxidase (HRP), and
chloramphenicol acetyltransferase (CAT). Additionally, epitope tags
are used in fusion protein constructions, including histidine (His)
tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G
tags, and thioredoxin (Trx) tags. Other fusion constructions can
include maltose binding protein (MBP), S-tag, Lex a DNA binding
domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes
simplex virus (HSV) BP16 protein fusions. A fusion protein also can
be engineered to contain a cleavage site located between the
HM74-like GPCR polypeptide-encoding sequence and the heterologous
protein sequence, so that the HM74-like GPCR polypeptide can be
cleaved and purified away from the heterologous moiety.
[0060] A fusion protein can be synthesized chemically, as is known
in the art. Preferably, a fusion protein is produced by covalently
linking two polypeptide segments or by standard procedures in the
art of molecular biology. Recombinant DNA methods can be used to
prepare fusion proteins, for example, by making a DNA construct
which comprises coding sequences selected from SEQ ID NO: 1 in
proper reading frame with nucleotides encoding the second
polypeptide segment and expressing the DNA construct in a host
cell, as is known in the art. Many kits for constructing fusion
proteins are available from companies such as Promega Corporation
(Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain
View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL
International Corporation (MIC; Watertown, Mass.), and Quantum
Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
[0061] Identification of Species Homologs
[0062] Species homologs of human M74-like GPCR polypeptide can be
obtained using HM74-like GPCR polynucleotides (described below) to
make suitable probes or primers for screening cDNA expression
libraries from other species, such as mice, monkeys, or yeast,
identifying cDNAs which encode homologs of HM74-like GPCR
polypeptide, and expressing the cDNAs as is known in the art.
[0063] Polynucleotides
[0064] An HM74-like GPCR polynucleotide can be single- or
double-stranded and comprises a coding sequence or the complement
of a coding sequence for an HM74-like GPCR polypeptide. A
nucleotide sequence encoding the HM74-like GPCR having SEQ ID NO: 2
is shown in SEQ ID NO: 1.
[0065] Degenerate nucleotide sequences encoding human HM74-like
GPCR polypeptides, as well as homologous nucleotide sequences which
are at least about 50, preferably about 75, 90, 96, or 98%
identical to the nucleotide sequence shown in SEQ ID NO: 1 or its
complement also are HM74-like GPCR polynucleotides. Percent
sequence identity between the sequences of two polynucleotides is
determined using computer programs such as ALIGN which employ the
FASTA algorithm, using an affine gap search with a gap open penalty
of -12 and a gap extension penalty of -2. Complementary DNA (cDNA)
molecules, species homologs, and variants of HM74-like GPCR
polynucleotides which encode biologically active HM74-like GPCR
polypeptides also are HM74-like GPCR polynucleotides.
[0066] Identification of Polynucleotide Variants and Homologs
[0067] Variants and homologs of the HM74-like GPCR polynucleotides
described above also are HM74-like GPCR polynucleotides. Typically,
homologous HM74-like GPCR polynucleotide sequences can be
identified by hybridization of candidate polynucleotides to known
HM74-like GPCR polynucleotides under stringent conditions, as is
known in the art. For example, using the following wash
conditions--2.times. SSC (0.3 M NaCl, 0.03 M sodium citrate, pH
7.0), 0.1% SDS, room temperature twice, 30 minutes each; then
2.times. SSC, 0.1% SDS, 50.degree. C. once, 30 minutes; then
2.times. SSC, room temperature twice, 10 minutes each--homologous
sequences can be identified which contain at most about 25-30%
basepair mismatches. More preferably, homologous nucleic acid
strands contain 15-25% basepair mismatches, even more preferably
5-15% basepair mismatches.
[0068] Species homologs of the HM74-like GPCR polynucleotides
disclosed herein also can be identified by making suitable probes
or primers and screening cDNA expression libraries from other
species, such as mice, monkeys, or yeast. Human variants of
HM74-like GPCR polynucleotides can be identified, for example, by
screening human cDNA expression libraries. It is well known that
the T.sub.m of a double-stranded DNA decreases by 1-1.5.degree. C.
with every 1% decrease in homology (Bonner et al., J. Mol. Biol.
81, 123 (1973). Variants of human HM74-like GPCR polynucleotides or
HM74-like GPCR polynucleotides of other species can therefore be
identified by hybridizing a putative homologous HM74-like GPCR
polynucleotide with a polynucleotide having a nucleotide sequence
of SEQ ID NO: 1 or the complement thereof to form a test hybrid.
The melting temperature of the test hybrid is compared with the
melting temperature of a hybrid comprising polynucleotides having
perfectly complementary nucleotide sequences, and the number or
percent of basepair mismatches within the test hybrid is
calculated.
[0069] Nucleotide sequences which hybridize to HM74-like GPCR
polynucleotides or their complements following stringent
hybridization and/or wash conditions also are HM74-like GPCR
polynucleotides. Stringent wash conditions are well known and
understood in the art and are disclosed, for example, in Sambrook
et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at
pages 9.50-9.51.
[0070] Typically, for stringent hybridization conditions a
combination of temperature and salt concentration should be chosen
that is approximately 12-20.degree. C. below the calculated T.sub.m
of the hybrid under study. The T.sub.m of a hybrid between an
HM74-like GPCR polynucleotide having a nucleotide sequence shown in
SEQ ID NO: 1 or the complement thereof and a polynucleotide
sequence which is at least about 50, preferably about 75, 90, 96,
or 98% identical to one of those nucleotide sequences can be
calculated, for example, using the equation of Bolton and McCarthy,
Proc. Natl. Acad. Sci U.S.A. 48, 1390 (1962):
T.sub.m=81.5.degree.
C.-16.6(log.sub.10[Na.sup.+])+0.41(%G+C)-0.63(%formam-
ide)-600/l),
[0071] where l=the length of the hybrid in basepairs.
[0072] Stringent wash conditions include, for example, 4.times. SSC
at 65.degree. C., or 50% formamide, 4.times. SSC at 42.degree. C.,
or 0.5.times. SSC, 0.1% SDS at 65.degree. C. Highly stringent wash
conditions include, for example, 0.2.times. SSC at 65.degree.
C.
[0073] Preparation of Polyinucleotides
[0074] An HM74-like GPCR polynucleotide can be isolated free of
other cellular components such as membrane components, proteins,
and lipids. Polynucleotides can be made by a cell and isolated
using standard nucleic acid purification techniques, or synthesized
using an amplification technique, such as the polymerase chain
reaction (PCR), or by using an automatic synthesizer. Methods for
isolating polynucleotides are routine and are known in the art. Any
such technique for obtaining a polynucleotide can be used to obtain
isolated HM74-like GPCR polynucleotides. For example, restriction
enzymes and probes can be used to isolate polynucleotide fragments
which comprises HM74-like GPCR nucleotide sequences. Isolated
polynucleotides are in preparations which are free or at least 70,
80, or 90% free of other molecules.
[0075] HM74-like GPCR cDNA molecules can be made with standard
molecular biology techniques, using HM74-like GPCR mRNA as a
template. HM74-like GPCR cDNA molecules can thereafter be
replicated using molecular biology techniques known in the art and
disclosed in manuals such as Sambrook et al. (1989). An
amplification technique, such as PCR, can be used to obtain
additional copies of polynucleotides of the invention, using either
human genomic DNA or cDNA as a template.
[0076] Alternatively, synthetic chemistry techniques can be used to
synthesizes HM.sup.74-like GPCR polynucleotides. The degeneracy of
the genetic code allows alternate nucleotide sequences to be
synthesized which will encode an HM74-like GPCR polypeptide having,
for example, an amino acid sequence shown in SEQ ID NO: 2 or a
biologically active variant thereof.
[0077] Extending Polynucleotides
[0078] Various PCR-based methods can be used to extend the nucleic
acid sequences encoding human HM74-like GPCR to detect upstream
sequences such as promoters and regulatory elements. For example,
restriction-site PCR uses universal primers to retrieve unknown
sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2,
318-322, 1993). Genomic DNA is first amplified in the presence of a
primer to a linker sequence and a primer specific to the known
region. The amplified sequences are then subjected to a second
round of PCR with the same linker primer and another specific
primer internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase.
[0079] Inverse PCR also can be used to amplify or extend sequences
using divergent primers based on a known region (Triglia et al.,
Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using
commercially available software, such as OLIGO 4.06 Primer Analysis
software (National Biosciences Inc., Plymouth, Minn.), to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to
anneal to the target sequence at temperatures about 68-72.degree.
C. The method uses several restriction enzymes to generate a
suitable fragment in the known region of a gene. The fragment is
then circularized by intramolecular ligation and used as a PCR
template.
[0080] Another method which can be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom
et al., PCR Methods Applic. 1, 111-119, 1991). In this method,
multiple restriction enzyme digestions and ligations also can be
used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR.
[0081] Another method which can be used to retrieve unknown
sequences is that of Parker et al., Nucleic Acids Res. 19,
3055-3060, 1991). Additionally, PCR, nested primers, and
PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used
to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process
avoids the need to screen libraries and is useful in finding
intron/exon junctions.
[0082] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Randomly-primed libraries are preferable, in that they will contain
more sequences which contain the 5' regions of genes. Use of a
randomly primed library may be especially preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries can be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0083] Commercially available capillary electrophoresis systems can
be used to analyze the size or confirm the nucleotide sequence of
PCR or sequencing products. For example, capillary sequencing can
employ flowable polymers for electrophoretic separation, four
different fluorescent dyes (one for each nucleotide) which are
laser activated, and detection of the emitted wavelengths by a
charge coupled device camera. Output/light intensity can be
converted to electrical signal using appropriate software (e.g.
GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire
process from loading of samples to computer analysis and electronic
data display can be computer controlled. Capillary electrophoresis
is especially preferable for the sequencing of small pieces of DNA
which might be present in limited amounts in a particular
sample.
[0084] Obtaining Polypeptides
[0085] HM74-like GPCR polypeptides can be obtained, for example, by
purification from human cells, by expression of HM74-like GPCR
polynucleotides, or by direct chemical synthesis.
[0086] Protein Purification
[0087] HM74-like GPCR polypeptides can be purified from any human
cell which expresses the receptor, including host cells which have
been transfected with HM74-like GPCR polynucleotides which express
such polypeptides. A purified HM74-like GPCR polypeptide is
separated from other compounds which normally associate with the
HM74-like GPCR polypeptide in the cell, such as certain proteins,
carbohydrates, or lipids, using methods well-known in the art. Such
methods include, but are not limited to, size exclusion
chromatography, ammonium sulfate fractionation, ion exchange
chromatography, affinity chromatography, and preparative gel
electrophoresis.
[0088] An HM74-like GPCR polypeptide can be conveniently isolated
as a complex with its associated G protein, as described in the
specific examples, below. A preparation of purified HM74-like GPCR
polypeptides is at least 80% pure; preferably, the preparations are
90%, 95%, or 99% pure. Purity of the preparations can be assessed
by any means known in the art, such as SDS-polyacrylamide gel
electrophoresis.
[0089] Expression of Polynucleotides
[0090] To express an HM74-like GPCR polypeptide, an HM74-like GPCR
polynucleotide can be inserted into an expression vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing sequences encoding HM74-like GPCR
polypeptides and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described, for example, in
Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
[0091] A variety of expression vector/host systems can be utilized
to contain and express sequences encoding an HM74-like GPCR
polypeptide. These include, but are not limited to, microorganisms,
such as bacteria transformed with recombinant bacteriophage,
plasmid, or cosmid DNA expression vectors; yeast transformed with
yeast expression vectors, insect cell systems infected with virus
expression vectors (e.g., baculovirus), plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids), or animal cell
systems.
[0092] The control elements or regulatory sequences are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements can vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, can be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1
plasmid (Life Technologies) and the like can be used. The
baculovirus polyhedrin promoter can be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO, and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) can be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferable. If it is
necessary to generate a cell line that contains--multiple copies of
a nucleotide sequence encoding an HM74-like GPCR polypeptide,
vectors based on SV40 or EBV can be used with an appropriate
selectable marker.
[0093] Bacterial and Yeast Expression Systems
[0094] In bacterial systems, a number of expression vectors can be
selected depending upon the use intended for the HM74-like GPCR
polypeptide. For example, when a large quantity of an HM74-like
GPCR polypeptide is needed for the induction of antibodies, vectors
which direct high level expression of fusion proteins that are
readily purified can be used. Such vectors include, but are not
limited to, multifunctional E. coli cloning and expression vectors
such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence
encoding the HM74-like GPCR polypeptide can be ligated into the
vector in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced. pIN vectors (Van Heeke & Schuster, J.
Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega,
Madison, Wis.) also can be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems can be designed to include heparin, thrombin, or factor Xa
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0095] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH can be used. For reviews, see
Ausubel et al. (1989) and Grant et al., Methods Enzymol. 153,
516-544, 1987.
[0096] Plant and Insect Expression Systems
[0097] If plant expression vectors are used, the expression of
sequences encoding HM74-like GPCR polypeptides can be driven by any
of a number of promoters. For example, viral promoters such as the
35S and 19S promoters of CaMV can be used alone or in combination
with the omega leader sequence from TMV (Takamatsu, EMBO J. 6,
307-311, 1987). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters can be used (Coruzzi et
al., EMBO J. 3, 1671-1680, 1984; Broglie et al., Science 224,
838-843, 1984; Winter et al., Results Probl. Cell Differ. 17,
85-105, 1991). These constructs can be introduced into plant cells
by direct DNA transformation or by pathogen-mediated transfection.
Such techniques are described in a number of generally available
reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE
AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196,
1992).
[0098] An insect system also can be used to express an HM74-like
GPCR polypeptide. For example, in one such system Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. Sequences encoding HM74-like GPCR polypeptides
can be cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin
promoter. Successful insertion of HM74-like GPCR polypeptides will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses can then be used to
infect S. frugiperda cells or Trichoplusia larvae in which
HM74-like GPCR polypeptides can be expressed (Engelhard et al.,
Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
[0099] Mammalian Expression Systems
[0100] A number of viral-based expression systems can be used to
express HM74-like GPCR polypeptides in mammalian host cells. For
example, if an adenovirus is used as an expression vector,
sequences encoding HM74-like GPCR polypeptides can be ligated into
an adenovirus transcription/translation complex comprising the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome can be used to
obtain a viable virus which is capable of expressing an HM74-like
GPCR polypeptide in infected host cells (Logan & Shenk, Proc.
Natl. Acad Sci. 81, 3655-3659, 1984). If desired, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be
used to increase expression in mammalian host cells.
[0101] Human artificial chromosomes (HACs) also can be used to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of 6M to 10M are constructed and delivered to
cells via conventional delivery methods (e.g., liposomes,
polycationic amino polymers, or vesicles).
[0102] Specific initiation signals also can be used to achieve more
efficient translation of sequences encoding HM74-like GPCR
polypeptides. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding an HM74-like
GPCR polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
(including the ATG initiation codon) should be provided. The
initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements
and initiation codons can be of various origins, both natural and
synthetic. The efficiency of expression can be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used (see Scharf et al., Results Probl. Cell
Differ. 20, 125-162, 1994).
[0103] Host Cells
[0104] A host cell strain can be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed HM74-like GPCR polypeptide in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the polypeptide also can be used to
facilitate correct insertion, folding and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC; 10801 University Boulevard,
Manassas, Va. 20110-2209) and can be chosen to ensure the correct
modification and processing of the foreign protein.
[0105] Stable expression is preferred for long-term, high-yield
production of recombinant proteins. For example, cell lines which
stably express HM74-like GPCR polypeptides can be transformed using
expression vectors which can contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells can be allowed to grow for 1-2 days in an
enriched medium before they are switched to a selective medium. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced HM74-like GPCR sequences.
Resistant clones of stably transformed cells can be proliferated
using tissue culture techniques appropriate to the cell type. See,
for example, ANIMAL CELL CULTURE, R. I. Freshney, ed., 1986.
[0106] Any number of selection systems can be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., Cell 11,
223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al.,
Cell 22, 817-23, 1980) genes which can be employed in tk.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For
example, dhfr confers resistance to methotrexate (Wigler et al.,
Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance
to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al.,
J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance
to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (Murray, 1992, supra). Additional selectable genes
have been described. For example, trpB allows cells to utilize
indole in place of tryptophan, or hisD, which allows cells to
utilize histinol in place of histidine (Hartman & Mulligan,
Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as
anthocyanins, .beta.-glucuronidase and its substrate GUS, and
luciferase and its substrate luciferin, can be used to identify
transformants and to quantify the amount of transient or stable
protein expression attributable to a specific vector system (Rhodes
et al., Methods Mol. Biol. 55, 121-131, 1995).
[0107] Detecting Expression of Polypeptides
[0108] Although the presence of marker gene expression suggests
that the HM74-like GPCR polynucleotide is also present, its
presence and expression may need to be confirmed. For example, if a
sequence encoding an HM74-like GPCR polypeptide is inserted within
a marker gene sequence, transformed cells containing sequences
which encode an HM74-like GPCR polypeptide can be identified by the
absence of marker gene function. Alternatively, a marker gene can
be placed in tandem with a sequence encoding an HM74-like GPCR
polypeptide under the control of a single promoter. Expression of
the marker gene in response to induction or selection usually
indicates expression of the HM74-like GPCR polynucleotide.
[0109] Alternatively, host cells which contain an HM74-like GPCR
polynucleotide and which express an HM74-like GPCR polypeptide can
be identified by a variety of procedures known to those of skill in
the art. These procedures include, but are not limited to, DNA-DNA
or DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip-based
technologies for the detection and/or quantification of nucleic
acid or protein. For example, the presence of a polynucleotide
sequence encoding an HM74-like GPCR polypeptide can be detected by
DNA-DNA or DNA-RNA hybridization or amplification using probes or
fragments or fragments of polynucleotides encoding an HM74-like
GPCR polypeptide. Nucleic acid amplification-based assays involve
the use of oligonucleotides selected from sequences encoding an
HM74-like GPCR polypeptide to detect transformants which contain an
HM74-like GPCR polynucleotide.
[0110] A variety of protocols for detecting and measuring the
expression of an HM74-like GPCR polypeptide, using either
polyclonal or monoclonal antibodies specific for the polypeptide,
are known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay using
monoclonal antibodies reactive to two non-interfering epitopes on
an HM74-like GPCR polypeptide can be used, or a competitive binding
assay can be employed. These and other assays are described in
Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS
Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158,
1211-1216, 1983).
[0111] A wide variety of labels and conjugation techniques are
known by those skilled in the art and can be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HM74-like GPCR polypeptides include
oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide. Alternatively, sequences encoding an
HM74-like GPCR polypeptide can be cloned into a vector for the
production of an mRNA probe. Such vectors are known in the art, are
commercially available, and can be used to synthesize RNA probes in
vitro by addition of labeled nucleotides and an appropriate RNA
polymerase such as T7, T3, or SP6. These procedures can be
conducted using a variety of commercially available kits (Amersham
Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter
molecules or labels which can be used for ease of detection include
radionuclides, enzymes, and fluorescent, chemiluminescent, or
chromogenic agents, as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0112] Expression and Purification of Polypeptides
[0113] Host cells transformed with nucleotide sequences encoding an
HM74-like GPCR polypeptide can be cultured under conditions
suitable for the expression and recovery of the protein from cell
culture. The polypeptide produced by a transformed cell can be
secreted or contained intracellularly depending on the sequence
and/or the vector used. As will be understood by those of skill in
the art, expression vectors containing polynucleotides which encode
HM74-like GPCR polypeptides can be designed to contain signal
sequences which direct secretion of soluble HM74-like GPCR
polypeptides through a prokaryotic or eukaryotic cell Membrane or
which direct the membrane insertion of membrane-bound HM74-like
GPCR polypeptide.
[0114] As discussed above, other constructions can be used to join
a sequence encoding an HM74-like GPCR polypeptide to a nucleotide
sequence encoding a polypeptide domain which will facilitate
purification of soluble proteins. Such purification facilitating
domains include, but are not limited to, metal chelating peptides
such as histidine-tryptophan modules that allow purification on
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). Inclusion of cleavable linker sequences such as those
specific for Factor Xa or enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and the HM74-like GPCR
polypeptide also can be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing an HM74-like GPCR polypeptide and 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification by IMAC (immobilized
metal ion affinity chromatography, as described in Porath et al.,
Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase
cleavage site provides a means for purifying the HM74-like GPCR
polypeptide from the fusion protein. Vectors which contain fusion
proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453,
1993.
[0115] Chemical Synthesis
[0116] Sequences encoding an HM74-like GPCR polypeptide can be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser.
215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232,
1980). Alternatively, an HM74-like GPCR polypeptide itself can be
produced using chemical methods to synthesize its amino acid
sequence, such as by direct peptide synthesis using solid-phase
techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963;
Roberge et al., Science 269, 202-204, 1995). Protein synthesis can
be performed using manual techniques or by automation. Automated
synthesis can be achieved, for example, using Applied Biosystems
431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of
HM74-like GPCR polypeptides can be separately synthesized and
combined using chemical methods to produce a full-length
molecule.
[0117] The newly synthesized peptide can be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, W H
Freeman and Co., New York, N.Y., 1983). The composition of a
synthetic HM74-like GPCR polypeptide can be confirmed by amino acid
analysis or sequencing (e.g., the Edman degradation procedure; see
Creighton, supra). Additionally, any portion of the amino acid
sequence of the HM74-like GPCR polypeptide can be altered during
direct synthesis and/or combined using chemical methods with
sequences from other proteins to produce a variant polypeptide or a
fusion protein.
[0118] Production of Altered Polypeptides
[0119] As will be understood by those of skill in the art, it may
be advantageous to produce HM74-like GPCR polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce an RNA transcript having desirable properties, such as a
half-life which is longer than that of a transcript generated from
the naturally occurring sequence.
[0120] The nucleotide sequences disclosed herein can be engineered
using methods generally known in the art to alter HM74-like GPCR
polypeptide-encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the polypeptide or mRNA product.
DNA shuffling by random fragmentation and PCR reassembly of gene
fragments and synthetic oligonucleotides can be used to engineer
the nucleotide sequences. For example, site-directed mutagenesis
can be used to insert new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants,
introduce mutations, and so forth.
[0121] Antibodies
[0122] Any type of antibody known in the art can be generated to
bind specifically to an epitope of an HM74-like GPCR polypeptide.
"Antibody" as used herein includes intact immunoglobulin molecules,
as well as fragments thereof, such as Fab, F(ab').sub.2, and Fv,
which are capable of binding an epitope of an HM74-like GPCR
polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino
acids are required to form an epitope. However, epitopes which
involve non-contiguous amino acids may require more, e.g., at least
15, 25, or 50 amino acids.
[0123] An antibody which specifically binds to an epitope of an
HM74-like GPCR polypeptide can be used therapeutically, as well as
in immunochemical assays, such as Western blots, ELISAs,
radioimmunoassays, immunohistochemical assays,
immunoprecipitations, or other immunochemical assays known in the
art. Various immunoassays can be used to identify antibodies having
the desired specificity. Numerous protocols for competitive binding
or immunoradiometric assays are well known in the art. Such
immunoassays typically involve the measurement of complex formation
between an immunogen and an antibody which specifically binds to
the immunogen.
[0124] Typically, an antibody which specifically binds to an
HM74-like GPCR polypeptide provides a detection signal at least 5-,
10-, or 20-fold higher than a detection signal provided with other
proteins when used in an immunochemical assay. Preferably,
antibodies which specifically bind to HM74-like GPCR polypeptides
do not detect other proteins in immunochemical assays and can
immunoprecipitate an HM74-like GPCR polypeptide from solution.
[0125] HM74-like GPCR polypeptides can be used to immunize a
mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human,
to produce polyclonal antibodies. If desired, an HM74-like GPCR
polypeptide can be conjugated to a carrier protein, such as bovine
serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
Depending on the host species, various adjuvants can be used to
increase the immunological response. Such adjuvants include, but
are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum
hydroxide), and surface active substances (e.g. lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, and dinitrophenol). Among adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum
are especially useful.
[0126] Monoclonal antibodies which specifically bind to an
HM74-like GPCR polypeptide can be prepared using any technique
which provides for the production of antibody molecules by
continuous cell lines in culture. These techniques include, but are
not limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler et al., Nature
256, 495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42,
1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole
et al., Mol. Cell Biol. 62, 109-120, 1984).
[0127] In addition, techniques developed for the production of
"chimeric antibodies," the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity, can be used (Morrison et al.,
Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al.,
Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454,
1985). Monoclonal and other antibodies also can be "humanized" to
prevent a patient from mounting an immune response against the
antibody when it is used therapeutically. Such antibodies may be
sufficiently similar in sequence to human antibodies to be used
directly in therapy or may require alteration of a few key
residues. Sequence differences between rodent antibodies and human
sequences can be minimized by replacing residues which differ from
those in the human sequences by site directed mutagenesis of
individual residues or by grating of entire complementarity
determining regions. Alternatively, humanized antibodies can be
produced using recombinant methods, as described in GB2188638B.
Antibodies which specifically bind to an HM74-like GPCR polypeptide
can contain antigen binding sites which are either partially or
fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
[0128] Alternatively, techniques described for the production of
single chain antibodies can be adapted using methods known in the
art to produce single chain antibodies which specifically bind to
HM74-like GPCR polypeptides. Antibodies with related specificity,
but of distinct idiotypic composition, can be generated by chain
shuffling from random combinatorial immunoglobin libraries (Burton,
Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
[0129] Single-chain antibodies also can be constructed using a DNA
amplification method, such as PCR, using hybridoma cDNA as a
template (Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11).
Single-chain antibodies can be mono- or bispecific, and can be
bivalent or tetravalent. Construction of tetravalent, bispecific
single-chain antibodies is taught, for example, in Coloma &
Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of
bivalent, bispecific single-chain antibodies is taught in Mallender
& Voss, 1994, J. Biol. Chem. 269, 199-206.
[0130] A nucleotide sequence encoding a single-chain antibody can
be constructed using manual or automated nucleotide synthesis,
cloned into an expression construct using standard recombinant DNA
methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology
(Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al.,
1993, J. Immunol. Meth. 165, 81-91).
[0131] Antibodies which specifically bind to HM74-like GPCR
polypeptides also can be produced by inducing in vivo production in
the lymphocyte population or by screening immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in the
literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837,
1989; Winter et al., Nature 349, 293-299, 1991).
[0132] Other types of antibodies can be constructed and used
therapeutically in methods of the invention. For example, chimeric
antibodies can be constructed as disclosed in WO 93/03151. Binding
proteins which are derived from immunoglobulins and which are
multivalent and multispecific, such as the "diabodies" described in
WO 94/13804, also can be prepared.
[0133] Antibodies according to the invention can be purified by
methods well known in the art. For example, antibodies can be
affinity purified by passage over a column to which an HM74-like
GPCR polypeptide is bound. The bound antibodies can then be eluted
from the column using a buffer with a high salt concentration.
[0134] Antisense Oligonucleotides
[0135] Antisense oligonucleotides are nucleotide sequences which
are complementary to a specific DNA or RNA sequence. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form complexes and block
either transcription or translation. Preferably, an antisense
oligonucleotide is at least 11 nucleotides in length, but can be at
least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides
long. Longer sequences also can be used. Antisense oligonucleotide
molecules can be provided in a DNA construct and introduced into a
cell as described above to decrease the level of HM74-like GPCR
gene products in the cell.
[0136] Antisense oligonucleotides can be deoxyribonucleotides,
ribonucleotides, or a combination of both. Oligonucleotides can be
synthesized manually or by an automated synthesizer, by covalently
linking the 5' end of one nucleotide with the 3' end of another
nucleotide with non-phosphodiester internucleotide linkages such
alkylphosphonates, phosphorothioates, phosphorodithioates,
alkylphosphonothioates, alkylphosphonates, phosphoramidates,
phosphate esters, carbamates, acetamidate, carboxymethyl esters,
carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol.
20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann
et al., Chem. Rev. 90, 543-583, 1990.
[0137] Modifications of HM74-like GPCR gene expression can be
obtained by designing antisense oligonucleotides which will form
duplexes to the control, 5' , or regulatory regions of the
HM74-like GPCR. Oligonucleotides derived from the transcription
initiation site, e.g., between positions -10 and +10 from the start
site, are preferred. Similarly, inhibition can be achieved using
"triple helix" base-pairing methodology. Triple helix pairing is
useful because it causes inhibition of the ability of the double
helix to open sufficiently for the binding of polymerases,
transcription factors, or chaperons. Therapeutic advances using
triplex DNA have been described in the literature (e.g., Gee et
al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES,
Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense
oligonucleotide also can be designed to block translation of mRNA
by preventing the transcript from binding to ribosomes.
[0138] Precise complementarity is not required for successful
complex formation between an antisense oligonucleotide and the
complementary sequence of an HM74-like GPCR polynucleotide.
Antisense oligonucleotides which comprise, for example, 2, 3, 4, or
5 or more stretches of contiguous nucleotides which are precisely
complementary to an HM74-like GPCR polynucleotide, each separated
by a stretch of contiguous nucleotides which are not complementary
to adjacent HM74-like GPCR nucleotides, can provide sufficient
targeting specificity for HM74-like GPCR mRNA. Preferably, each
stretch of complementary contiguous nucleotides is at least 4, 5,
6, 7, or 8 or more nucleotides in length. Non-complementary
intervening sequences are preferably 1, 2, 3, or 4 nucleotides in
length. One skilled in the art can easily use the calculated
melting point of an antisense-sense pair to determine the degree of
mismatching which will be tolerated between a particular antisense
oligonucleotide and a particular HM74-like GPCR polynucleotide
sequence.
[0139] Antisense oligonucleotides can be modified without affecting
their ability to hybridize to an HM74-like GPCR polynucleotide.
These modifications can be internal or at one or both ends of the
antisense molecule. For example, internucleoside phosphate linkages
can be modified by adding cholesteryl or diamine moieties with
varying numbers of carbon residues between the amino groups and
terminal ribose. Modified bases and/or sugars, such as arabinose
instead of ribose, or a 3', 5'-substituted oligo nucleotide in
which the 3' hydroxyl group or the 5' phosphate group are
substituted, also can be employed in a modified antisense
oligonucleotide. These modified oligonucleotides can be prepared by
methods well known in the art. See, e.g., Agrawal et al., Trends
Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90,
543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542,
1987.
[0140] Ribozymes
[0141] Ribozymes are RNA molecules with catalytic activity. See,
e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem.
59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609;
1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996.
Ribozymes can be used to inhibit gene function by cleaving an RNA
sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat.
No. 5,641,673). The mechanism of ribozyme action involves
sequence-specific hybridization of the ribozyme molecule to
complementary target RNA, followed by endonucleolytic cleavage.
Examples include engineered hammerhead motif ribozyme molecules
that can specifically and efficiently catalyze endonucleolytic
cleavage of specific nucleotide sequences.
[0142] The coding sequence of an HM74-like GPCR polynucleotide can
be used to generate ribozymes which will specifically bind to mRNA
transcribed from the HM74-like GPCR polynucleotide. Methods of
designing and constructing ribozymes which can cleave other RNA
molecules in trans in a highly sequence specific manner have been
developed and described in the art (see Haseloff et al. Nature 334,
585-591, 1988). For example, the cleavage activity of ribozymes can
be targeted to specific RNAs by engineering a discrete
"hybridization" region into the ribozyme. The hybridization region
contains a sequence complementary to the target RNA and thus
specifically hybridizes with the target (see, for example, Gerlach
et al., EP 321,201).
[0143] Specific ribozyme cleavage sites within an HM74-like GPCR
RNA target can be identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
RNA containing the cleavage site can be evaluated for secondary
structural features which may render the target inoperable.
Suitability of candidate HM74-like GPCR RNA targets also can be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays. Longer complementary sequences can be used to increase the
affinity of the hybridization sequence for the target. The
hybridizing and cleavage regions of the ribozyme can be integrally
related such that upon hybridizing to the target RNA through the
complementary regions, the catalytic region of the ribozyme can
cleave the target.
[0144] Ribozymes can be introduced into cells as part of a DNA
construct. Mechanical methods, such as microinjection,
liposome-mediated transfection, electroporation, or calcium
phosphate precipitation, can be used to introduce a
ribozyme-containing DNA construct into cells in which it is desired
to decrease HM74-like GPCR expression. Alternatively, if it is
desired that the cells stably retain the DNA construct, the
construct can be supplied on a plasmid and maintained as a separate
element or integrated into the genome of the cells, as is known in
the art. A ribozyme-encoding DNA construct can include
transcriptional regulatory elements, such as a promoter element, an
enhancer or UAS element, and a transcriptional terminator signal,
for controlling transcription of ribozymes in the cells.
[0145] As taught in Haseloff et al., U.S. Pat. No. 5,641,673,
ribozymes can be engineered so that ribozyme expression will occur
in response to factors which induce expression of a target gene.
Ribozymes also can be engineered to provide an additional level of
regulation, so that destruction of mRNA occurs only when both a
ribozyme and a target gene are induced in the cells.
[0146] Screening Methods
[0147] The invention provides assays for screening test compounds
which bind to and/or modulate the activity of an HM74-like GPCR
polypeptide or an HM74-like GPCR polynucleotide. A test compound
preferably binds to an HM74-like GPCR polypeptide or
polynucleotide. More preferably, a test compound decreases or
increases a biological effect mediated via human HM74-like GPCR by
at least about 10, preferably about 50, more preferably about 75,
90, or 100% relative to the absence of the test compound.
[0148] Test Compounds
[0149] Test compounds can be pharmacologic agents already known in
the art or can be compounds previously unknown to have any
pharmacological activity. The compounds can be naturally occurring
or designed in the laboratory. They can be isolated from
microorganisms, animals, or plants, and can be produced
recombinantly, or synthesized by chemical methods known in the art.
If desired, test compounds can be obtained using any of the
numerous combinatorial library methods known in the art, including
but not limited to, biological libraries, spatially addressable
parallel solid phase or solution phase libraries, synthetic library
methods requiring deconvolution, the "one-bead one-compound"
library method, and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to polypeptide libraries, while the other four approaches
are applicable to polypeptide, non-peptide oligomer, or small
molecule libraries of compounds. See Lam, Anticancer Drug Des. 12,
145, 1997.
[0150] Methods for the synthesis of molecular libraries are well
known in the art (see, for example, DeWitt et al., Proc. Natl.
Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci.
U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678,
1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew.
Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem.
Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233,
1994). Libraries of compounds can be presented in solution (see,
e.g., Houghten, Biotechniques 13, 412-421, 1992), or on beads (Lam,
Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993),
bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992),
or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin,
Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad. Sci
97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and
Ladner, U.S. Pat. No. 5,223,409).
[0151] High Throughput Screening
[0152] Test compounds can be screened for the ability to bind to
HM74-like GPCR polypeptides or polynucleotides or to affect
HM74-like GPCR activity or HM74-like GPCR gene expression using
high throughput screening. Using high throughput screening, many
discrete compounds can be tested in parallel so that large numbers
of test compounds can be quickly screened. The most widely
established techniques utilize 96-well microtiter plates. The wells
of the microtiter plates typically require assay volumes that range
from 50 to 500 .mu.l. In addition to the plates, many instruments,
materials, pipettors, robotics, plate washers, and plate readers
are commercially available to fit the 96-well format.
[0153] Alternatively, "free format assays," or assays that have no
physical barrier between samples, can be used. For example, an
assay using pigment cells (melanocytes) in a simple homogeneous
assay for combinatorial peptide libraries is described by
Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18
(1994). The cells are placed under agarose in petri dishes, then
beads that carry combinatorial compounds are placed on the surface
of the agarose. The combinatorial compounds are partially released
the compounds from the beads. Active compounds can be visualized as
dark pigment areas because, as the compounds diffuse locally into
the gel matrix, the active compounds cause the cells to change
colors.
[0154] Another example of a free format assay is described by
Chelsky, "Strategies for Screening Combinatorial Libraries: Novel
and Traditional Approaches," reported at the First Annual
Conference of The Society for Biomolecular Screening in
Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple
homogenous enzyme assay for carbonic anhydrase inside an agarose
gel such that the enzyme in the gel would cause a color change
throughout the gel. Thereafter, beads carrying combinatorial
compounds via a photolinker were placed inside the gel and the
compounds were partially released by UV-light. Compounds that
inhibited the enzyme were observed as local zones of inhibition
having less color change.
[0155] Yet another example is described by Salmon et al., Molecular
Diversity 2, 57-63 (1996). In this example, combinatorial libraries
were screened for compounds that had cytotoxic effects on cancer
cells growing in agar.
[0156] Another high throughput screening method is described in
Beutel et al., U.S. Pat. No. 5,976,813. In this method, test
samples are placed in a porous matrix. One or more assay components
are then placed within, on top of, or at the bottom of a matrix
such as a gel, a plastic sheet, a filter, or other form of easily
manipulated solid support. When samples are introduced to the
porous matrix they diffuse sufficiently slowly, such that the
assays can be performed without the test samples running
together.
[0157] Binding Assays
[0158] For binding assays, the test compound is preferably a small
molecule which binds to and occupies the active site of the
HM74-like GPCR polypeptide, thereby making the ligand binding site
inaccessible to substrate such that normal biological activity is
prevented. Examples of such small molecules include, but are not
limited to, small peptides or peptide-like molecules. Potential
ligands which may bind to a polypeptide of the invention include,
but are not limited to, the natural ligands of known GPCRs and
analogues or derivatives thereof. Natural ligands of GPCRs include,
for example, adrenomedullin, amylin, calcitonin gene related
protein (CGRP), calcitonin, anandamide, serotonin, histamine,
adrenalin, noradrenalin, platelet activating factor, thrombin, C5a,
bradykinin, and chemokines.
[0159] In binding assays, either the test compound or the HM74-like
GPCR polypeptide can comprise a detectable label, such as a
fluorescent, radioisotopic, chemiluminescent, or enzymatic label,
such as horseradish peroxidase, alkaline phosphatase, or
luciferase. Detection of a test compound which is bound to the
HM74-like GPCR polypeptide can then be accomplished, for example,
by direct counting of radioemmission, by scintillation counting, or
by determining conversion of an appropriate substrate to a
detectable product.
[0160] Alternatively, binding of a test compound to an HM74-like
GPCR polypeptide can be determined without labeling either of the
interactants. For example, a microphysiometer can be used to detect
binding of a test compound with an HM74-like GPCR polypeptide. A
microphysiometer (e.g., Cytosensor.TM.) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a test compound and an HM74-like GPCR
polypeptide (McConnell et al., Science 257, 1906-1912, 1992).
[0161] Determining the ability of a test compound to bind to an
HM74-like GPCR polypeptide also can be accomplished using a
technology such as real-time Bimolecular Interaction Analysis (BIA)
(Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and
Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a
technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[0162] In yet another aspect of the invention, an HM74-like GPCR
polypeptide can be used as a "bait protein" in a two-hybrid assay
or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos
et al., Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268,
12046-12054, 1993; Bartel et al., Biotechniques 14, 920-924, 1993;
Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and Brent
W094/10300), to identify other proteins which bind to or interact
with the HM74-like GPCR polypeptide and modulate its activity.
[0163] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. For example, in one construct, polynucleotide encoding
an HM74-like GPCR polypeptide can be fused to a polynucleotide
encoding the DNA binding domain of a known transcription factor
(e.g., GAL-4). In the other construct a DNA sequence that encodes
an unidentified protein ("prey" or "sample") can be fused to a
polynucleotide that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact in vivo to form an protein-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription
of a reporter gene (e.g., LacZ), which is operably linked to a
transcriptional regulatory site responsive to the transcription
factor. Expression of the reporter gene can be detected, and cell
colonies containing the functional transcription factor can be
isolated and used to obtain the DNA sequence encoding the protein
which interacts with the HM74-like GPCR polypeptide.
[0164] It may be desirable to immobilize either the HM74-like GPCR
polypeptide (or polynucleotide) or the test compound to facilitate
separation of bound from unbound forms of one or both of the
interactants, as well as to accommodate automation of the assay.
Thus, either the HM74-like GPCR polypeptide (or polynucleotide) or
the test compound can be bound to a solid support. Suitable solid
supports include, but are not limited to, glass or plastic slides,
tissue culture plates, microtiter wells, tubes, silicon chips, or
particles such as beads (including, but not limited to, latex,
polystyrene, or glass beads). Any method known in the art can be
used to attach the HM74-like GPCR polypeptide (or polynucleotide)
or test compound to a solid support, including use of covalent and
non-covalent linkages, passive absorption, or pairs of binding
moieties attached respectively to the polypeptide (or
polynucleotide) or test compound and the solid support. Test
compounds are preferably bound to the solid support in an array, so
that the location of individual test compounds can be tracked.
Binding of a test compound to an HM74-like GPCR polypeptide (or
polynucleotide) can be accomplished in any vessel suitable for
containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and microcentrifuge tubes.
[0165] In one embodiment, the HM74-like GPCR polypeptide is a
fusion protein comprising a domain that allows the HM74-like GPCR
polypeptide to be bound to a solid support. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical) or glutathione
derivatized microtiter plates, which are then combined with the
test compound or the test compound and the non-adsorbed HM74-like
GPCR polypeptide; the mixture is then incubated under conditions
conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads or microtiter
plate wells are washed to remove any unbound components. Binding of
the interactants can be determined either directly or indirectly,
as described above. Alternatively, the complexes can be dissociated
from the solid support before binding is determined.
[0166] Other techniques for immobilizing proteins or
polynucleotides on a solid support also can be used in the
screening assays of the invention. For example, either an HM74-like
GPCR polypeptide (or polynucleotide) or a test compound can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated HM74-like GPCR polypeptides (or polynucleotides) or
test compounds can be prepared from biotin-NHS(-hydroxysuccinimide)
using techniques well known in the art (e.g., biotinylation kit,
Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies which specifically bind to an HM74-like
GPCR polypeptide, polynucleotide, or a test compound, but which do
not interfere with a desired binding site, such as the active site
of the HM74-like GPCR polypeptide, can be derivatized to the wells
of the plate. Unbound target or protein can be trapped in the wells
by antibody conjugation.
[0167] Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies which specifically
bind to the HM74-like GPCR polypeptide or test compound,
enzyme-linked assays which rely on detecting an activity of the
HM74-like GPCR polypeptide, and SDS gel electrophoresis under
non-reducing conditions.
[0168] Screening for test compounds which bind to an HM74-like GPCR
polypeptide or polynucleotide also can be carried out in an intact
cell. Any cell which comprises an HM74-like GPCR polypeptide or
polynucleotide can be used in a cell-based assay system. An
HM74-like GPCR polynucleotide can be naturally occurring in the
cell or can be introduced using techniques such as those described
above. Binding of the test compound to an HM74-like GPCR
polypeptide or polynucleotide is determined as described above.
[0169] Functional Assays
[0170] Test compounds can be tested for the ability to increase or
decrease a biological effect of an HM74-like GPCR polypeptide. Such
biological effects can be determined using functional assays such
as those described in the specific examples, below. Functional
assays can be carried out after contacting either a purified
HM74-like GPCR polypeptide, a cell membrane preparation, or an
intact cell with a test compound. A test compound which decreases
an activity of an HM74-like GPCR by at least about 10, preferably
about 50, more preferably about 75, 90, or 100% is identified as a
potential agent for decreasing HM74-like GPCR activity. A test
compound which increases an HM74-like GPCR activity by at least
about 10, preferably about 50, more preferably about 75, 90, or
100% is identified as a potential agent for increasing HM74-like
GPCR activity.
[0171] One such screening procedure involves the use of
melanophores which are transfected to express an HM74-like GPCR
polypeptide. Such a screening technique is described in WO 92/01810
published Feb. 6, 1992. Thus, for example, such an assay may be
employed for screening for a compound which inhibits activation of
the receptor polypeptide by contacting the melanophore cells which
comprise the receptor with both a receptor ligand and a test
compound to be screened. Inhibition of the signal generated by the
ligand indicates that a test compound is a potential antagonist for
the receptor, i e, inhibits activation of the receptor. The screen
may be employed for identifying a test compound which activates the
receptor by contacting such cells with compounds to be screened and
determining whether each test compound generates a signal, i.e.,
activates the receptor.
[0172] Other screening techniques include the use of cells which
express a human HM74-like GPCR polypeptide (for example,
transfected CHO cells) in a system which measures extracellular pH
changes caused by receptor activation (see, e.g., Science 246,
181-296, 1989). For example, test compounds may be contacted with a
cell which expresses a human HM74-like GPCR polypeptide and a
second messenger response, e.g., signal transduction or pH changes,
can be measured to determine whether the test compound activates or
inhibits the receptor.
[0173] Another such screening technique involves introducing RNA
encoding a human HM74-like GPCR polypeptide into Xenopus oocytes to
transiently express the receptor. The transfected oocytes can then
be contacted with the receptor ligand and a test compound to be
screened, followed by detection of inhibition or activation of a
calcium signal in the case of screening for test compounds which
are thought to inhibit activation of the receptor.
[0174] Another screening technique involves expressing a human
HM74-like GPCR polypeptide in cells in which the receptor is linked
to a phospholipase C or D. Such cells include endothelial cells,
smooth muscle cells, embryonic kidney cells, etc. The screening may
be accomplished as described above by quantifying the degree of
activation of the receptor from changes in the phospholipase
activity.
[0175] Details of functional assays such as those described above
are provided in the specific examples, below.
[0176] Gene Expression
[0177] In another embodiment, test compounds which increase or
decrease HM74-like GPCR gene expression are identified. An
HM74-like GPCR polynucleotide is contacted with a test compound,
and the expression of an RNA or polypeptide product of the
HM74-like GPCR polynucleotide is determined. The level of
expression of appropriate mRNA or polypeptide in the presence of
the test compound is compared to the level of expression of mRNA or
polypeptide in the absence of the test compound. The test compound
can then be identified as a modulator of expression based on this
comparison. For example, when expression of mRNA or polypeptide is
greater in the presence of the test compound than in its absence,
the test compound is identified as a stimulator or enhancer of the
mRNA or polypeptide expression. Alternatively, when expression of
the mRNA or polypeptide is less in the presence of the test
compound than in its absence, the test compound is identified as an
inhibitor of the mRNA or polypeptide expression.
[0178] The level of HM74-like GPCR mRNA or polypeptide expression
in the cells can be determined by methods well known in the art for
detecting mRNA or polypeptide. Either qualitative or quantitative
methods can be used. The presence of polypeptide products of an
HM74-like GPCR polynucleotide can be determined, for example, using
a variety of techniques known in the art, including immunochemical
methods such as radioimmunoassay, Western blotting, and
immunohistochemistry. Alternatively, polypeptide synthesis can be
determined in vivo, in a cell culture, or in an in vitro
translation system by detecting incorporation of labeled amino
acids into an HM74-like GPCR polypeptide.
[0179] Such screening can be carried out either in a cell-free
assay system or in an intact cell. Any cell which expresses an
HM74-like GPCR polynucleotide can be used in a cell-based assay
system. The HM74-like GPCR polynucleotide can be naturally
occurring in the cell or can be introduced using techniques such as
those described above. Either a primary culture or an established
cell line, such as CHO or human embryonic kidney 293 cells, can be
used.
[0180] Pharmaceutical Compositions
[0181] The invention also provides pharmaceutical compositions
which can be administered to a patient to achieve a therapeutic
effect. Pharmaceutical compositions of the invention can comprise,
for example, an HM74-like GPCR polypeptide, HM.sup.74-like GPCR
polynucleotide, antibodies which specifically bind to an HM74-like
GPCR polypeptide, or mimetics, agonists, antagonists, or inhibitors
of an HM74-like GPCR polypeptide activity. The compositions can be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which can be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions can be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0182] In addition to the active ingredients, these pharmaceutical
compositions can contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Pharmaceutical compositions of the invention
can be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, parenteral, topical,
sublingual, or rectal means. Pharmaceutical compositions for oral
administration can be formulated using pharmaceutically acceptable
carriers well known in the art in dosages suitable for oral
administration. Such carriers enable the pharmaceutical
compositions to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
[0183] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents can
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0184] Dragee cores can be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which also can
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments can be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0185] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds can be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0186] Pharmaceutical formulations suitable for parenteral
administration can be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions can contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds can be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic
amino polymers also can be used for delivery. Optionally, the
suspension also can contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. For topical or nasal
administration, penetrants appropriate to the particular barrier to
be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0187] The pharmaceutical compositions of the present invention can
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes. The pharmaceutical composition can be
provided as a salt and can be formed with many acids, including but
not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric,
malic, succinic, etc. Salts tend to be more soluble in aqueous or
other protonic solvents than are the corresponding free base forms.
In other cases, the preferred preparation can be a lyophilized
powder which can contain any or all of the following: 1-50 mM
histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5
to 5.5, that is combined with buffer prior to use.
[0188] Further details on techniques for formulation and
administration can be found in the latest edition of REMINGTON'S
PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After
pharmaceutical compositions have been prepared, they can be placed
in an appropriate container and labeled for treatment of an
indicated condition. Such labeling would include amount, frequency,
and method of administration.
[0189] Therapeutic Indications and Methods
[0190] GPCRs are ubiquitous in the mammalian host and are
responsible for many biological functions, including many
pathologies. Accordingly, it is desirable to find compounds and
drugs which stimulate a GPCR on the one hand and which can inhibit
the function of a GPCR on the other hand. For example, compounds
which activate a GPCR may be employed for therapeutic purposes,
such as the treatment of asthma, inflammation, CNS disorders,
including Parkinson's disease, acute heart failure, urinary
retention, and osteoporosis. In particular, compounds which
activate GPCRs are useful in treating various cardiovascular
ailments such as caused by the lack of pulmonary blood flow or
hypertension. In addition these compounds may also be used in
treating various physiological disorders relating to abnormal
control of fluid and electrolyte homeostasis and in diseases
associated with abnormal angiotensin-induced aldosterone
secretion.
[0191] In general, compounds which inhibit activation of a GPCR can
be used for a variety of therapeutic purposes, for example, for the
treatment of hypotension and/or hypertension, angina pectoris,
myocardial infarction, inflammation, ulcers, asthma, allergies,
benign prostatic hypertrophy, and psychotic and neurological
disorders including schizophrenia, manic excitement, depression,
delirium, dementia or severe mental retardation, dyskinesias, such
as Huntington's disease or Tourett's syndrome, among others.
Compounds which inhibit GPCRs also are useful in reversing
endogenous anorexia and in the control of bulimia.
[0192] This invention further pertains to the use of novel agents
identified by the screening assays described above. Accordingly, it
is within the scope of this invention to use a test compound
identified as described herein in an appropriate animal model. For
example, an agent identified as described herein (e.g., a
modulating agent, an antisense nucleic acid molecule, a specific
antibody, ribozyme, or an HM74-like GPCR polypeptide binding
molecule) can be used in an animal model to determine the efficacy,
toxicity, or side effects of treatment with such an agent.
Alternatively, an agent identified as described herein can be used
in an animal model to determine the mechanism of action of such an
agent. Furthermore, this invention pertains to uses of novel agents
identified by the above-described screening assays for treatments
as described herein.
[0193] A reagent which affects HM74-like GPCR activity can be
administered to a human cell, either in vitro or in vivo, to reduce
HM74-like GPCR activity. The reagent preferably binds to an
expression product of a human HM74-like GPCR gene. If the
expression product is a protein, the reagent is preferably an
antibody. For treatment of human cells ex vivo, an antibody can be
added to a preparation of stem cells which have been removed from
the body. The cells can then be replaced in the same or another
human body, with or without clonal propagation, as is known in the
art.
[0194] In one embodiment, the reagent is delivered using a
liposome. Preferably, the liposome is stable in the animal into
which it has been administered for at least about 30 minutes, more
preferably for at least about 1 hour, and even more preferably for
at least about 24 hours. A liposome comprises a lipid composition
that is capable of targeting a reagent, particularly a
polynucleotide, to a particular site in an animal, such as a human.
Preferably, the lipid composition of the liposome is capable of
targeting to a specific organ of an animal, such as the lung,
liver, spleen, heart brain, lymph nodes, and skin.
[0195] A liposome useful in the present invention comprises a lipid
composition that is capable of fusing with the plasma membrane of
the targeted cell to deliver its contents to the cell. Preferably,
the transfection efficiency of a liposome is about 0.5 .mu.g of DNA
per 16 nmole of liposome delivered to about 106' cells, more
preferably about 1.0 .mu.g of DNA per 16 nmole of liposome
delivered to about 10 cells, and even more preferably about 2.0
.mu.g of DNA per 16 nmol of liposome delivered to about 106 cells.
Preferably, a liposome is between about 100 and 500 nm, more
preferably between about 150 and 450 nm, and even more preferably
between about 200 and 400 nm in diameter.
[0196] Suitable liposomes for use in the present invention include
those liposomes standardly used in, for example, gene delivery
methods known to those of skill in the art. More preferred
liposomes include liposomes having a polycationic lipid composition
and/or liposomes having a cholesterol backbone conjugated to
polyethylene glycol. Optionally, a liposome comprises a compound
capable of targeting the liposome to a particular cell types, such
as a cell-specific ligand exposed on the outer surface of the
liposome.
[0197] Complexing a liposome with a reagent such as an antisense
oligonucleotide or ribozyme can be achieved using methods which are
standard in the art (see, for example, U.S. Pat. No. 5,705,151).
Preferably, from about 0.1 .mu.g to about 10 .mu.g of
polynucleotide is combined with about 8 nmol of liposomes, more
preferably from about 0.5 .mu.g to about 5 .mu.g of polynucleotides
are combined with about 8 nmol liposomes, and even more preferably
about 1.0 .mu.g of polynucleotides is combined with about 8 nmol
liposomes.
[0198] In another embodiment, antibodies can be delivered to
specific tissues in vivo using receptor-mediated targeted delivery.
Receptor-mediated DNA delivery techniques are taught in, for
example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993);
Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT
GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, J. Biol.
Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46
(1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59
(1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).
[0199] Determination of a Therapeutically Effective Dose
[0200] The determination of a therapeutically effective dose is
well within the capability of those skilled in the art. A
therapeutically effective dose refers to that amount of active
ingredient which increases or decreases HM74-like GPCR activity
relative to the HM74-like GPCR activity which occurs in the absence
of the therapeutically effective dose.
[0201] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model also
can be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0202] Therapeutic efficacy and toxicity, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population), can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals. The dose ratio of toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the ratio,
LD.sub.50/ED.sub.50.
[0203] Pharmaceutical compositions which exhibit large therapeutic
indices are preferred. The data obtained from cell culture assays
and animal studies is used in formulating a range of dosage for
human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include the
ED.sub.50 with little or no toxicity. The dosage varies within this
range depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0204] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active ingredient or to maintain the desired effect. Factors
which can be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions can be
administered every 3 to 4 days, every week, or once every two weeks
depending on the half-life and clearance rate of the particular
formulation.
[0205] Normal dosage amounts can vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0206] If the reagent is a single-chain antibody, polynucleotides
encoding the antibody can be constructed and introduced into a cell
either ex vivo or in vivo using well-established techniques
including, but not limited to, transferrin-polycation-mediated DNA
transfer, transfection with naked or encapsulated nucleic acids,
liposome-mediated cellular fusion, intracellular transportation of
DNA-coated latex beads, protoplast fusion, viral infection,
electroporation, "gene gun," and DEAE- or calcium
phosphate-mediated transfection.
[0207] Effective in vivo dosages of an antibody are in the range of
about 5 .mu.g to about 50 .mu.g/kg, about 50 .mu.g to about 5
mg/kg, about 100 .mu.g to about 500 .mu.g/kg of patient body
weight, and about 200 to about 250 .mu.g/kg of patient body weight.
For administration of polynucleotides encoding single-chain
antibodies, effective in vivo dosages are in the range of about 100
ng to about 200 ng, 500 ng to about 50 mg, about 1 .mu.g to about 2
mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about
100 .mu.g of DNA.
[0208] If the expression product is mRNA, the reagent is preferably
an antisense oligonucleotide or a ribozyme. Polynucleotides which
express antisense oligonucleotides or ribozymes can be introduced
into cells by a variety of methods, as described above.
[0209] Preferably, a reagent reduces expression of an HM74-like
GPCR gene or the activity of an HM74-like GPCR polypeptide by at
least about 10, preferably about 50, more preferably about 75, 90,
or 100% relative to the absence of the reagent. The effectiveness
of the mechanism chosen to decrease the level of expression of an
HM74-like GPCR gene or the activity of an HM74-like GPCR
polypeptide can be assessed using methods well known in the art,
such as hybridization of nucleotide probes to HM74like
GPCR-specific mRNA, quantitative RT-PCR, immunologic detection of
an HM74-like GPCR polypeptide, or measurement of HM74-like GPCR
activity.
[0210] In any of the embodiments described above, any of the
pharmaceutical compositions of the invention can be administered in
combination with other appropriate therapeutic agents. Selection of
the appropriate agents for use in combination therapy can be made
by one of ordinary skill in the art, according to conventional
pharmaceutical principles. The combination of therapeutic agents
can act synergistically to effect the treatment or prevention of
the various disorders described above. Using this approach, one may
be able to achieve therapeutic efficacy with lower dosages of each
agent, thus reducing the potential for adverse side effects.
[0211] Any of the therapeutic methods described above can be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0212] Diagnostic Methods
[0213] GPCRs also can be used in diagnostic assays for detecting
diseases and abnormalities or susceptibility to diseases and
abnormalities related to the presence of mutations in the nucleic
acid sequences which encode a GPCR. Such diseases, by way of
example, are related to cell transformation, such as tumors and
cancers, and various cardiovascular disorders, including
hypertension and hypotension, as well as diseases arising from
abnormal blood flow, abnormal angiotensin-induced aldosterone
secretion, and other abnormal control of fluid and electrolyte
homeostasis.
[0214] According to the present invention, differences can be
determined between the cDNA or genomic sequence encoding HM74-like
GPCR in individuals afflicted with a disease and in normal
individuals. If a mutation is observed in some or all of the
afflicted individuals but not in normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0215] Sequence differences between a reference gene and a gene
having mutations can be revealed by the direct DNA sequencing
method. In addition, cloned DNA segments can be employed as probes
to detect specific DNA segments. The sensitivity of this method is
greatly enhanced when combined with PCR. For example, a sequencing
primer can be used with a double-stranded PCR product or a
single-stranded template molecule generated by a modified PCR. The
sequence determination is performed by conventional procedures
using radiolabeled nucleotides or by automatic sequencing
procedures using fluorescent tags.
[0216] Genetic testing based on DNA sequence differences can be
carried out by detection of alteration in electrophoretic mobility
of DNA fragments in gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized, for example,
by high resolution gel electrophoresis. DNA fragments of different
sequences can be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science 230, 1242, 1985). Sequence changes at specific
locations can also be revealed by nuclease protection assays, such
as RNase and S1 protection or the chemical cleavage method (e.g.,
Cotton et al., Proc. Natl. Acad. Sci. USA 485, 4397-4401, 1985).
Thus, the detection of a specific DNA sequence can be performed by
methods such as hybridization, RNase protection, chemical cleavage,
direct DNA sequencing or the use of restriction enzymes and
Southern blotting of genomic DNA. In addition to direct methods
such as gel-electrophoresis and DNA sequencing, mutations can also
be detected by in situ analysis.
[0217] Altered levels of an HM74-like GPCR also can be detected in
various tissues. Assays used to detect levels of the receptor
polypeptides in a body sample, such as blood or a tissue biopsy,
derived from a host are well known to those of skill in the art and
include radioimmunoassays, competitive binding assays, Western blot
analysis, and ELISA assays.
[0218] All patents and patent applications cited in this disclosure
are expressly incorporated herein by reference. The above
disclosure generally describes the present invention. A more
complete understanding can be obtained by reference to the
following specific examples which are provided for purposes of
illustration only and are not intended to limit the scope of the
invention.
EXAMPLE 1
[0219] Detection of HM74-Like GPCR Activity
[0220] The polynucleotide of SEQ ID NO: 1 is inserted into the
expression vector pCEV4 and the expression vector pCEV4-HM74-like
GPCR polypeptide obtained is transfected into human embryonic
kidney 293 cells. The cells are scraped from a culture flask into 5
ml of Tris HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell
lysates are centrifuged at 1000 rpm for 5 minutes at 4.degree. C.
The supernatant is centrifuged at 30,000.times.g for 20 minutes at
4.degree. C. The pellet is suspended in binding buffer containing
50 mM Tris HCl, 5 mM MgSO.sub.4, 1 mM EDTA, 100 mM NaCl, pH 7.5,
supplemented with 0.1% BSA, 2 .mu.g/ml aprotinin, 0.5 mg/ml
leupeptin, and 10 .mu.g/ml phosphoramidon. Optimal membrane
suspension dilutions, defined as the protein concentration required
to bind less than 10% of an added radioligand, i.e.
.sup.125I-labeled HM74, are added to 96-well polypropylene
microtiter plates containing ligand, non-labeled peptides, and
binding buffer to a final volume of 250 .mu.l.
[0221] In equilibrium saturation binding assays, membrane
preparations are incubated in the presence of increasing
concentrations (0.1 nM to 4 nM) of 1251 ligand.
[0222] Binding reaction mixtures are incubated for one hour at
30.degree. C. The reaction is stopped by filtration through GF/B
filters treated with 0.5% polyethyleneimine, using a cell
harvester. Radioactivity is measured by scintillation counting, and
data are analyzed by a computerized non-linear regression program.
Non-specific binding is defined as the amount of radioactivity
remaining after incubation of membrane protein in the presence of
100 nM of unlabeled peptide. Protein concentration is measured by
the Bradford method using Bio-Rad Reagent, with bovine serum
albumin as a standard. The HM74-like GPCR activity of the
polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is
demonstrated.
EXAMPLE 2
[0223] Radioligand Binding Assays
[0224] Human embryonic kidney 293 cells transfected with a
polynucleotide which expresses human HM74-like GPCR are scraped
from a culture flask into 5 ml of Tris HCl, 5 mM EDTA, pH 7.5, and
lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5
minutes at 4.degree. C. The supernatant is centrifuged at
30,000.times.g for 20 minutes at 4.degree. C. The pellet is
suspended in binding buffer containing 50 mM Tris HCl, 5 mM
MgSO.sub.4, 1 mM EDTA, 100 mM NaCl, pH 7.5, supplemented with 0.1%
BSA, 2 .mu.g/ml aprotinin, 0.5 mg/ml leupeptin, and 10 .mu.g/ml
phosphoramidon. Optimal membrane suspension dilutions, defined as
the protein concentration required to bind less than 10% of the
added radioligand, i.e. HM74, are added to 96-well polypropylene
microtiter plates containing .sup.125I-labeled ligand or test
compound, non-labeled peptides, and binding buffer to a final
volume of 250 .mu.l.
[0225] In equilibrium saturation binding assays, membrane
preparations are incubated in the presence of increasing
concentrations (0.1 nM to 4 nM) of .sup.125I-labeled ligand or test
compound (specific activity 2200 Ci/mmol). The binding affinities
of different test compounds are determined in equilibrium
competition binding assays, using 0.1 nM .sup.125I-peptide in the
presence of twelve different concentrations of each test
compound.
[0226] Binding reaction mixtures are incubated for one hour at
30.degree. C. The reaction is stopped by filtration through GF/B
filters treated with 0.5% polyethyleneimine, using a cell
harvester. Radioactivity is measured by scintillation counting, and
data are analyzed by a computerized non-linear regression
program.
[0227] Non-specific binding is defined as the amount of
radioactivity remaining after incubation of membrane protein in the
presence of 100 nM of unlabeled peptide. Protein concentration is
measured by the Bradford method using Bio-Rad Reagent, with bovine
serum albumin as a standard. A test compound which increases the
radioactivity of membrane protein by at least 15% relative to
radioactivity of membrane protein which was not incubated with a
test compound is identified as a compound which binds to a human
HM74-like GPCR polypeptide.
EXAMPLE3
[0228] Effect of a Test Compound on Human HM74-Like GPCR-Mediated
Cyclic AMP Formation
[0229] Receptor-mediated inhibition of cAMP formation can be
assayed in host cells which express human HM74-like GPCR. Cells are
plated in 96-well plates and incubated in Dulbecco's phosphate
buffered saline (PBS) supplemented with 10 mM HEPES, 5 mM
theophylline, 2 .mu.g/ml aprotinin, 0.5 mg/ml leupeptin, and 10
.mu.g/ml phosphoramidon for 20 minutes at 37.degree. C. in 5%
CO.sub.2. A test compound is added and incubated for an additional
10 minutes at 37.degree. C. The medium is aspirated, and the
reaction is stopped by the addition of 100 mM HCl. The plates are
stored at 4.degree. C. for 15 minutes. cAMP content in the stopping
solution is measured by radioimmunoassay.
[0230] Radioactivity is quantified using a gamma counter equipped
with data reduction software. A test compound which decreases
radioactivity of the contents of a well relative to radioactivity
of the contents of a well in the absence of the test compound is
identified as a potential inhibitor of cAMP formation. A test
compound which increases radioactivity of the contents of a well
relative to radioactivity of the contents of a well in the absence
of the test compound is identified as a potential enhancer of cAMP
formation.
EXAMPLE 4
[0231] Effect of a Test Compound on the Mobilization of
Intracellular Calcium
[0232] Intracellular free calcium concentration can be measured by
microspectrofluorometry using the fluorescent indicator dye
Fura-2/AM (Bush et al., J. Neurochem. 57, 562-74, 1991). Stably
transfected cells are seeded onto a 35 mm culture dish containing a
glass coverslip insert. Cells are washed with HBS, incubated with a
test compound, and loaded with 100 .mu.l of Fura-2/AM (10 .mu.M)
for 20-40 minutes. After washing with HBS to remove the Fura-2/AM
solution, cells are equilibrated in HBS for 10-20 minutes. Cells
are then visualized under the 40.times. objective of a Leitz
Fluovert FS microscope.
[0233] Fluorescence emission is determined at 510 nM, with
excitation wavelengths alternating between 340 nM and 380 nM. Raw
fluorescence data are converted to calcium concentrations using
standard calcium concentration curves and software analysis
techniques. A test compound which increases the fluorescence by at
least 15% relative to fluorescence in the absence of a test
compound is identified as a compound which mobilizes intracellular
calcium.
EXAMPLE 5
[0234] Effect of a Test Compound on Phosphoinositide Metabolism
[0235] Cells which stably express human HM74-like GPCR cDNA are
plated in 96-well plates and grown to confluence. The day before
the assay, the growth medium is changed to 100 .mu.l of medium
containing 1% serum and 0.5 .mu.Ci .sup.3H-myinositol. The plates
are incubated overnight in a CO.sub.2 incubator (5% CO.sub.2 at
37.degree. C.). Immediately before the assay, the medium is removed
and replaced by 200 .mu.l of PBS containing 10 mM LiCl, and the
cells are equilibrated with the new medium for 20 minutes.
[0236] During this interval, cells also are equilibrated with
antagonist, added as a 10 .mu.l aliquot of a 20-fold concentrated
solution in PBS.
[0237] The .sup.3H-inositol phosphate accumulation from inositol
phospholipid metabolism is started by adding 10 .mu.l of a solution
containing a test compound. To the first well 10 .mu.l are added to
measure basal accumulation. Eleven different concentrations of test
compound are assayed in the following 11 wells of each plate row.
All assays are performed in duplicate by repeating the same
additions in two consecutive plate rows.
[0238] The plates are incubated in a CO.sub.2 incubator for one
hour. The reaction is terminated by adding 15 .mu.l of 50% v/v
trichloroacetic acid (TCA), followed by a 40 minute incubation at
4.degree. C. After neutralizing TCA with 40 .mu.l of 1 M Tris, the
content of the wells is transferred to a Multiscreen HV filter
plate (Millipore) containing Dowex AG1-X8 (200-400 mesh, formate
form). The filter plates are prepared by adding 200 .mu.l of Dowex
AG1-X8 suspension (50% v/v, water:resin) to each well. The filter
plates are placed on a vacuum manifold to wash or elute the resin
bed. Each well is washed 2 times with 200 .mu.l of water, followed
by 2.times.200 .mu.l of 5 mM sodium tetraborate/60 mM ammonium
formate.
[0239] The .sup.3H-IPs are eluted into empty 96-well plates with
200 .mu.l of 1.2 M ammonium formate/0.1 formic acid. The content of
the wells is added to 3 ml of scintillation cocktail, and
radioactivity is determined by liquid scintillation counting.
EXAMPLE 6
[0240] Receptor Binding Methods
[0241] Standard Binding Assays. Binding assays are carried out in a
binding buffer containing 50 mM HEPES, pH 7.4, 0.5% BSA, and 5 mM
MgCl.sub.2. The standard assay for radioligand (e.g.,
.sup.125I-test compound) binding to membrane fragments comprising
HM74-like GPCR polypeptides is carried out as follows in 96 well
microtiter plates (e.g., Dynatech Immulon II Removawell plates).
Radioligand is diluted in binding buffer+PMSF/Baci to the desired
cpm per 50 .mu.l, then 50 .mu.l aliquots are added to the wells.
For non-specific binding samples, 5 .mu.l of 40 .mu.M cold ligand
also is added per well. Binding is initiated by adding 150 .mu.l
per well of membrane diluted to the desired concentration (10-30
.mu.g membrane protein/well) in binding buffer+PMSF/Baci. Plates
are then covered with Linbro mylar plate sealers (Flow Labs) and
placed on a Dynatech Microshaker II. Binding is allowed to proceed
at room temperature for 1-2 hours and is stopped by centrifuging
the plate for 15 minutes at 2,000.times.g. The supernatants are
decanted, and the membrane pellets are washed once by addition of
200 .mu.l of ice cold binding buffer, brief shaking, and
recentrifugation. The individual wells are placed in 12.times.75 mm
tubes and counted in an LKB Gammamaster counter (78% efficiency).
Specific binding by this method is identical to that measured when
free ligand is removed by rapid (3-5 seconds) filtration and
washing on polyethyleneimine-coated glass fiber filters.
[0242] Three variations of the standard binding assay are also
used.
[0243] 1. Competitive radioligand binding assays with a
concentration range of cold ligand vs. .sup.125 I-labeled ligand
are carried out as described above with one modification. All
dilutions of ligands being assayed are made in 40.times. PMSF/Baci
to a concentration 40.times. the final concentration in the assay.
Samples of peptide (5 .mu.l each) are then added per microtiter
well. Membranes and radioligand are diluted in binding buffer
without protease inhibitors. Radioligand is added and mixed with
cold ligand, and then binding is initiated by addition of
membranes.
[0244] 2. Chemical cross-linking of radioligand with receptor is
done after a binding step identical to the standard assay. However,
the wash step is done with binding buffer minus BSA to reduce the
possibility of non-specific cross-linking of radioligand with BSA.
The cross-linking step is carried out as described below.
[0245] 3. Larger scale binding assays to obtain membrane pellets
for studies on solubilization of receptor:ligand complex and for
receptor purification are also carried out. These are identical to
the standard assays except that (a) binding is carried out in
polypropylene tubes in volumes from 1-250 ml, (b) concentration of
membrane protein is always 0.5 mg/ml, and (c) for receptor
purification, BSA concentration in the binding buffer is reduced to
0.25%, and the wash step is done with binding buffer without BSA,
which reduces BSA contamination of the purified receptor.
EXAMPLE 7
[0246] Chemical Cross-Linking of Radioligand to Receptor
[0247] After a radioligand binding step as described above,
membrane pellets are resuspended in 200 .mu.l per microtiter plate
well of ice-cold binding buffer without BSA. Then 5 .mu.l per well
of 4 mM N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS, Pierce) in
DMSO is added and mixed. The samples are held on ice and
UV-irradiated for 10 minutes with a Mineralight R-52G lamp (UVP
Inc., San Gabriel, Calif.) at a distance of 5-10 cm. Then the
samples are transferred to Eppendorf microfuge tubes, the membranes
pelleted by centrifugation, supernatants removed, and membranes
solubilized in Laemmli SDS sample buffer for polyacrylamide gel
electrophoresis (PAGE). PAGE is carried out as described below.
Radiolabeled proteins are visualized by autoradiography of the
dried gels with Kodak XAR film and Dupont image intensifier
screens.
EXAMPLE 8
[0248] Membrane Solubilization
[0249] Membrane solubilization is carried out in buffer containing
25 mM Tris, pH 8, 10% glycerol (w/v) and 0.2 mM CaCl.sub.2
(solubilization buffer). The highly soluble detergents including
Triton X-100, deoxycholate, deoxycholate:lysolecithin, CHAPS, and
zwittergent are made up in solubilization buffer at 10%
concentrations and stored as frozen aliquots. Lysolecithin is made
up fresh because of insolubility upon freeze-thawing and digitonin
is made fresh at lower concentrations due to its more limited
solubility.
[0250] To solubilize membranes, washed pellets after the binding
step are resuspended free of visible particles by pipetting and
vortexing in solubilization buffer at 100,000.times.g for 30
minutes. The supernatants are removed and held on ice and the
pellets are discarded.
EXAMPLE 9
[0251] Assay of Solubilized Receptors
[0252] After binding of .sup.125I ligands and solubilization of the
membranes with detergent, the intact R:L complex can be assayed by
four different methods. All are carried out on ice or in a cold
room at 4-10.degree. C.).
[0253] 1. Column chromatography (Knuhtsen et al., Biochem. J. 254,
641-647, 1988). Sephadex G-50 columns (8.times.250 mm) are
equilibrated with solubilization buffer containing detergent at the
concentration used to solubilize membranes and 1 mg/ml bovine serum
albumin. Samples of solubilized membranes (0.2-0.5 ml) are applied
to the columns and eluted at a flow rate of about 0.7 ml/minute.
Samples (0.18 ml) are collected. Radioactivity is determined in a
gamma counter. Void volumes of the columns are determined by the
elution volume of blue dextran. Radioactivity eluting in the void
volume is considered bound to protein. Radioactivity eluting later,
at the same volume as free .sup.125I ligands, is considered
non-bound.
[0254] 2. Polyethyleneglycol precipitation (Cuatrecasas, Proc.
Natl. Acad. Sci. USA 69, 318-322, 1972). For a 100 .mu.l sample of
solubilized membranes in a 12.times.75 mm polypropylene tube, 0.5
ml of 1% (w/v) bovine gamma globulin (Sigma) in 0.1 M sodium
phosphate buffer is added, followed by 0.5 ml of 25% (w/v)
polyethyleneglycol (Sigma) and mixing. The mixture is held on ice
for 15 minutes. Then 3 ml of 0.1 M sodium phosphate, pH 7.4, is
added per sample. The samples are rapidly (1-3 seconds) filtered
over Whatman GF/B glass fiber filters and washed with 4 ml of the
phosphate buffer. PEG-precipitated receptor: .sup.125I-ligand
complex is determined by gamma counting of the filters.
[0255] 3. GFB/PEI filter binding (Bruns et al., Analytical Biochem.
132, 74-81, 1983). Whatman GF/B glass fiber filters are soaked in
0.3% polyethyleneimine (PEI, Sigma) for 3 hours. Samples of
solubilized membranes (25-100 .mu.l) are replaced in 12.times.75 mm
polypropylene tubes. Then 4 ml of solubilization buffer without
detergent is added per sample and the samples are immediately
filtered through the GFB/PEI filters (1-3 seconds) and washed with
4 ml of solubilization buffer. CPM of receptor: .sup.125I-ligand
complex adsorbed to filters are determined by gamma counting.
[0256] 4. Charcoal/Dextran (Paul and Said, Peptides 7[Suppl.
1],147-149, 1986). Dextran T70 (0.5 g, Pharmacia) is dissolved in 1
liter of water, then 5 g of activated charcoal (Norit A, alkaline;
Fisher Scientific) is added. The suspension is stirred for 10
minutes at room temperature and then stored at 4.degree. C. until
use. To measure R:L complex, 4 parts by volume of charcoal/dextran
suspension are added to 1 part by volume of solubilized membrane.
The samples are mixed and held on ice for 2 minutes and then
centrifuged for 2 minutes at 11,000.times.g in a Beckman microfuge.
Free radioligand is adsorbed charcoal/dextran and is discarded with
the pellet. Receptor: .sup.125I-ligand complexes remain in the
supernatant and are determined by gamma counting.
EXAMPLE 10
[0257] Receptor Purification
[0258] Binding of biotinyl-receptor to GH.sub.4 C1 membranes is
carried out as described above. Incubations are for 1 hour at room
temperature. In the standard purification protocol, the binding
incubations contain 10 nM Bio-S29. .sup.125I ligand is added as a
tracer at levels of 5,000-100,000 cpm per mg of membrane protein.
Control incubations contain 10 .mu.M cold ligand to saturate the
receptor with non-biotinylated ligand.
[0259] Solubilization of receptor:ligand complex also is carried
out as described above, with 0.15% deoxycholate: lysolecithin in
solubilization buffer containing 0.2 mM MgCl.sub.2, to obtain
100,000.times.g supernatants containing solubilized R:L
complex.
[0260] Immobilized streptavidin (streptavidin cross-linked to 6%
beaded agarose, Pierce Chemical Co.; "SA-agarose") is washed in
solubilization buffer and added to the solubilized membranes as
{fraction (1/30)} of the final volume. This mixture is incubated
with constant stirring by end-over-end rotation for 4-5 hours at
4-10.degree. C. Then the mixture is applied to a column and the
non-bound material is washed through. Binding of radioligand to
SA-agarose is determined by comparing cpm in the 100,000.times.g
supernatant with that in the column effluent after adsorption to
SA-agarose. Finally, the column is washed with 12-15 column volumes
of solubilization buffer+0.15% deoxycholate:lysolecithin+{fractio-
n (1/500)} (vol/vol) 100.times.4 pase.
[0261] The streptavidin column is eluted with solubilization
buffer+0.1 mM EDTA+0.1 mM EGTA+0.1 mM GTP-gamma-S (Sigma)+0.15%
(wt/vol) deoxycholate:lysolecithin+{fraction (1/1000)} (vol/vol)
100.times.4 pase. First, one column volume of elution buffer is
passed through the column and flow is stopped for 20-30 minutes.
Then 3-4 more column volumes of elution buffer are passed through.
All the eluates are pooled.
[0262] Eluates from the streptavidin column are incubated overnight
(12-15 hours) with immobilized wheat germ agglutinin (WGA agarose,
Vector Labs) to adsorb the receptor via interaction of covalently
bound carbohydrate with the WGA lectin. The ratio (vol/vol) of
WGA-agarose to streptavidin column eluate is generally 1:400. A
range from 1:1000 to 1:200 also can be used. After the binding
step, the resin is pelleted by centrifugation, the supernatant is
removed and saved, and the resin is washed 3 times (about 2 minutes
each) in buffer containing 50 mM HEPES, pH 8, 5 mM MgCl.sub.2, and
0.15% deoxycholate:lysolecithin. To elute the WGA-bound receptor,
the resin is extracted three times by repeated mixing (vortex mixer
on low speed) over a 15-30 minute period on ice, with 3 resin
columns each time, of 10 mM N-N'-N"-tri-acetylchitotriose in the
same HEPES buffer used to wash the resin. After each elution step,
the resin is centrifuged down and the supernatant is carefully
removed, free of WGA-agarose pellets. The three, pooled eluates
contain the final, purified receptor. The material non-bound to WGA
contain G protein subunits specifically eluted from the
streptavidin column, as well as non-specific contaminants. All
these fractions are stored frozen at -90.degree. C.
EXAMPLE 11
[0263] Identification of Test Compounds That Bind to HM74-Like GPCR
Polypeptides
[0264] Purified HM74-like GPCR polypeptides comprising a
glutathione-S-transferase protein and absorbed onto
glutathione-derivatized wells of 96-well microtiter plates are
contacted with test compounds from a small molecule library at pH
7.0 in a physiological buffer solution. HM74-like GPCR polypeptides
comprise an amino acid sequence shown in SEQ ID NO: 2. The test
compounds comprise a fluorescent tag.
[0265] The samples are incubated for 5 minutes to one hour. Control
samples are incubated in the absence of a test compound.
[0266] The buffer solution containing the test compounds is washed
from the wells. Binding of a test compound to an HM74-like GPCR
polypeptide is detected by fluorescence measurements of the
contents of the wells. A test compound which increases the
fluorescence in a well by at least 15% relative to fluorescence of
a well in which a test compound was not incubated is identified as
a compound which binds to an HM74-like GPCR polypeptide.
EXAMPLE 12
[0267] Identification of a Test Compound Which Decreases HM74-Like
GPCR Gene Expression
[0268] A test compound is administered to a culture of human
gastric cells and incubated at 37.degree. C. for 10 to 45 minutes.
A culture of the same type of cells incubated for the same time
without the test compound provides a negative control.
[0269] RNA is isolated from the two cultures as described in
Chirgwin et al., Biochem. 18, 5294-99, 1979). Northern blots are
prepared using 20 to 30 .mu.g total RNA and hybridized with a
.sup.32P-labeled HM74-like GPCR-specific probe at 65.degree. C. in
Express-hyb (CLONTECH). The probe comprises at least 11 contiguous
nucleotides selected from the complement of SEQ ID NO: 1. A test
compound which decreases the HM74-like GPCR-specific signal
relative to the signal obtained in the absence of the test compound
is identified as an inhibitor of HM74-like GPCR gene
expression.
EXAMPLE 13
[0270] Treatment of Asthma With a Reagent Which Specifically Binds
to an HM74-Like GPCR Gene Product
[0271] Synthesis of antisense HM74-like GPCR oligonucleotides
comprising at least 11 contiguous nucleotides selected from the
complement of SEQ ID NO: 1 is performed on a Pharmacia Gene
Assembler series synthesizer using the phosphoramidite procedure
(Uhlmann et al., Chem. Rev. 90, 534-83, 1990). Following assembly
and deprotection, oligonucleotides are ethanol-precipitated twice,
dried, and suspended in phosphate-buffered saline (PBS) at the
desired concentration. Purity of these oligonucleotides is tested
by capillary gel electrophoreses and ion exchange HPLC. Endotoxin
levels in the oligonucleotide preparation are determined using the
Luminous Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.)
105, 361-362, 1953).
[0272] The antisense oligonucleotides are administered
intrabronchially to a patient with asthma. The severity of the
patient's asthma is decreased.
Sequence CWU 1
1
4 1 1038 DNA Homo sapiens 1 atgtacaacg ggtcgtgctg ccgcatcgag
ggggacacca tctcccaggt gatgccgccg 60 ctgctcattg tggcctttgt
gctgggcgca ctaggcaatg gggtcgccct gtgtggtttc 120 tgcttccaca
tgaagacctg gaagcccagc actgtttacc ttttcaattt ggccgtggct 180
gatttcctcc ttatgatctg cctgcctttt cggacagact attacctcag acgtagacac
240 tgggcttttg gggacattcc ctgccgagtg gggctcttca cgttggccat
gaacagggcc 300 gggagcatcg tgttccttac ggtggtggct gcggacaggt
atttcaaagt ggtccacccc 360 caccacgcgg tgaacactat ctccacccgg
gtggcggctg gcatcgtctg caccctgtgg 420 gccctggtca tcctgggaac
agtgtatctt ttgctggaga accatctctg cgtgcaagag 480 acggccgtct
cctgtgagag cttcatcatg gagtcggcca atggctggca tgacatcatg 540
ttccagctgg agttctttat gcccctcggc atcatcttat tttgctcctt caagattgtt
600 tggagcctga ggcggaggca gcagctggcc agacaggctc ggatgaagaa
ggcgacccgg 660 ttcatcatgg tggtggcaat tgtgttcatc acatgctacc
tgcccagcgt gtctgctaga 720 ctctatttcc tctggacggt gccctcgagt
gcctgcgatc cctctgtcca tggggccctg 780 cacataaccc tcagcttcac
ctacatgaac agcatgctgg atcccctggt gtattatttt 840 tcaagcccct
cctttcccaa attctacaac aagctcaaaa tctgcagtct gaaacccaag 900
cagccaggac actcaaaaac acaaaggccg gaagagatgc caatttcgaa cctcggtcgc
960 aggagttgca tcagtgtggc aaatagtttc caaagccagt ctgatgggca
atgggatccc 1020 cacattgttg agtggcac 1038 2 346 PRT Homo sapiens 2
Met Tyr Asn Gly Ser Cys Cys Arg Ile Glu Gly Asp Thr Ile Ser Gln 1 5
10 15 Val Met Pro Pro Leu Leu Ile Val Ala Phe Val Leu Gly Ala Leu
Gly 20 25 30 Asn Gly Val Ala Leu Cys Gly Phe Cys Phe His Met Lys
Thr Trp Lys 35 40 45 Pro Ser Thr Val Tyr Leu Phe Asn Leu Ala Val
Ala Asp Phe Leu Leu 50 55 60 Met Ile Cys Leu Pro Phe Arg Thr Asp
Tyr Tyr Leu Arg Arg Arg His 65 70 75 80 Trp Ala Phe Gly Asp Ile Pro
Cys Arg Val Gly Leu Phe Thr Leu Ala 85 90 95 Met Asn Arg Ala Gly
Ser Ile Val Phe Leu Thr Val Val Ala Ala Asp 100 105 110 Arg Tyr Phe
Lys Val Val His Pro His His Ala Val Asn Thr Ile Ser 115 120 125 Thr
Arg Val Ala Ala Gly Ile Val Cys Thr Leu Trp Ala Leu Val Ile 130 135
140 Leu Gly Thr Val Tyr Leu Leu Leu Glu Asn His Leu Cys Val Gln Glu
145 150 155 160 Thr Ala Val Ser Cys Glu Ser Phe Ile Met Glu Ser Ala
Asn Gly Trp 165 170 175 His Asp Ile Met Phe Gln Leu Glu Phe Phe Met
Pro Leu Gly Ile Ile 180 185 190 Leu Phe Cys Ser Phe Lys Ile Val Trp
Ser Leu Arg Arg Arg Gln Gln 195 200 205 Leu Ala Arg Gln Ala Arg Met
Lys Lys Ala Thr Arg Phe Ile Met Val 210 215 220 Val Ala Ile Val Phe
Ile Thr Cys Tyr Leu Pro Ser Val Ser Ala Arg 225 230 235 240 Leu Tyr
Phe Leu Trp Thr Val Pro Ser Ser Ala Cys Asp Pro Ser Val 245 250 255
His Gly Ala Leu His Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met 260
265 270 Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Lys
Phe 275 280 285 Tyr Asn Lys Leu Lys Ile Cys Ser Leu Lys Pro Lys Gln
Pro Gly His 290 295 300 Ser Lys Thr Gln Arg Pro Glu Glu Met Pro Ile
Ser Asn Leu Gly Arg 305 310 315 320 Arg Ser Cys Ile Ser Val Ala Asn
Ser Phe Gln Ser Gln Ser Asp Gly 325 330 335 Gln Trp Asp Pro His Ile
Val Glu Trp His 340 345 3 1730 DNA Homo sapiens 3 ttcttcttgc
cttctgggca gctttgtgtg ctctccagca cggagtccga ggaggaacag 60
cgaagctgaa aatgaaaatt actctggcgc tggacccaat cgctcctcta cggcatcaat
120 ctcatcggac ccccccaccc taccgcctct cagaaatgac cacttttgca
aaattgcatg 180 catttccaag cttcatccgg ctccaggctt ggcctctccc
agaggcaggc ggcttgtgag 240 acgggctcca gagaaaggac ctccctgggt
ctctcatttc ctggctgaag tttctcttct 300 cgctgctgtg gcagcatcca
acccacacac acaggacccg catcctgggt gatgaagtca 360 gacacgcagc
agctgggtga gtgctaacgc tcagataagc atctgtgcca ttgtggggac 420
tccctgggct gctctgcacc cggacacttg ctctgtcccc gccatgtaca acgggtcgtg
480 ctgccgcatc gagggggaca ccatctccca ggtgatgccg ccgctgctca
ttgtggcctt 540 tgtgctgggc gcactaggca atggggtcgc cctgtgtggt
ttctgcttcc acatgaagac 600 ctggaagccc agcactgttt accttttcaa
tttggccgtg gctgatttcc tccttatgat 660 ctgcctgcct tttcggacag
actattacct cagacgtaga cactgggctt ttggggacat 720 tccctgccga
gtggggctct tcacgttggc catgaacagg gccgggagca tcgtgttcct 780
tacggtggtg gctgcggaca ggtatttcaa agtggtccac ccccaccacg cggtgaacac
840 tatctccacc cgggtggcgg ctggcatcgt ctgcaccctg tgggccctgg
tcatcctggg 900 aacagtgtat cttttgctgg agaaccatct ctgcgtgcaa
gagacggccg tctcctgtga 960 gagcttcatc atggagtcgg ccaatggctg
gcatgacatc atgttccagc tggagttctt 1020 tatgcccctc ggcatcatct
tattttgctc cttcaagatt gtttggagcc tgaggcggag 1080 gcagcagctg
gccagacagg ctcggatgaa gaaggcgacc cggttcatca tggtggtggc 1140
aattgtgttc atcacatgct acctgcccag cgtgtctgct agactctatt tcctctggac
1200 ggtgccctcg agtgcctgcg atccctctgt ccatggggcc ctgcacataa
ccctcagctt 1260 cacctacatg aacagcatgc tggatcccct ggtgtattat
ttttcaagcc cctcctttcc 1320 caaattctac aacaagctca aaatctgcag
tctgaaaccc aagcagccag gacactcaaa 1380 aacacaaagg ccggaagaga
tgccaatttc gaacctcggt cgcaggagtt gcatcagtgt 1440 ggcaaatagt
ttccaaagcc agtctgatgg gcaatgggat ccccacattg ttgagtggca 1500
ctgaacaagc agaccaacaa cactgaggaa gatagagtgg tgacttagaa ttaactcgtg
1560 ctaaggggtc gggggctttg aaaatgccac ccccctttct tattgcaaga
cggcttctcg 1620 cacatgaact gcatccttct cattctgtcg gaaatgaaat
tcacacaact ataccttttg 1680 gggaggttcc agttgattga agtgagttgg
ctgcattttc ttatctgatc 1730 4 387 PRT Homo sapiens 4 Met Asn Arg His
His Leu Gln Asp His Phe Leu Glu Ile Asp Lys Lys 1 5 10 15 Asn Cys
Cys Val Phe Arg Asp Asp Phe Ile Ala Lys Val Leu Pro Pro 20 25 30
Val Leu Gly Leu Glu Phe Ile Phe Gly Leu Leu Gly Asn Gly Leu Ala 35
40 45 Leu Trp Ile Phe Cys Phe His Leu Lys Ser Trp Lys Ser Ser Arg
Ile 50 55 60 Phe Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu Ile
Ile Cys Leu 65 70 75 80 Pro Phe Val Met Asp Tyr Tyr Val Arg Arg Ser
Asp Trp Asn Phe Gly 85 90 95 Asp Ile Pro Cys Arg Leu Val Leu Phe
Met Phe Ala Met Asn Arg Gln 100 105 110 Gly Ser Ile Ile Phe Leu Thr
Val Val Ala Val Asp Arg Tyr Phe Arg 115 120 125 Val Val His Pro His
His Ala Leu Asn Lys Ile Ser Asn Trp Thr Ala 130 135 140 Ala Ile Ile
Ser Cys Leu Leu Trp Gly Ile Thr Val Gly Leu Thr Val 145 150 155 160
His Leu Leu Lys Lys Lys Leu Leu Ile Gln Asn Gly Pro Ala Asn Val 165
170 175 Cys Ile Ser Phe Ser Ile Cys His Thr Phe Arg Trp His Glu Ala
Met 180 185 190 Phe Leu Leu Glu Phe Leu Leu Pro Leu Gly Ile Ile Leu
Phe Cys Ser 195 200 205 Ala Arg Ile Ile Trp Ser Leu Arg Gln Arg Gln
Met Asp Arg His Ala 210 215 220 Lys Ile Lys Arg Ala Ile Thr Phe Ile
Met Val Val Ala Ile Val Phe 225 230 235 240 Val Ile Cys Phe Leu Pro
Ser Val Val Val Arg Ile Arg Ile Phe Trp 245 250 255 Leu Leu His Thr
Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg Ser Val 260 265 270 Asp Leu
Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met 275 280 285
Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Asn Phe 290
295 300 Phe Ser Thr Leu Ile Asn Arg Cys Leu Gln Arg Lys Met Thr Gly
Glu 305 310 315 320 Pro Asp Asn Asn Arg Ser Thr Ser Val Glu Leu Thr
Gly Asp Pro Asn 325 330 335 Lys Thr Arg Gly Ala Pro Glu Ala Leu Met
Ala Asn Ser Gly Glu Pro 340 345 350 Trp Ser Pro Ser Tyr Leu Gly Pro
Thr Ser Asn Asn His Ser Lys Lys 355 360 365 Gly His Cys His Gln Glu
Pro Ala Ser Leu Glu Lys Gln Leu Gly Cys 370 375 380 Cys Ile Glu
385
* * * * *